Induced Resistance for Plant Defense (Walters/Induced Resistance for Plant Defense) || Mechanisms of Defence to Pathogens: Biochemistry and Physiology

Trim Size: 170mm x 244mm Walter c06.tex V3 - 07/22/2014 9:22 A.M. Page 106

6 Mechanisms of Defence to Pathogens:Biochemistry and Physiology

Christophe Garcion1∗, Olivier Lamotte2∗, Jean-Luc Cacas3∗

and Jean-Pierre Métraux4

1INRA, Univ. Bordeaux, Villenave d’Ornon, France2CNRS, Pôle Mécanismes et gestion des interactions Plantes-Micro-organismes, Dijon,France3Université de Bourgogne, UMR Agroecologie, Pôle Mécanismes et gestion desinteractions Plantes-Micro-organismes, Dijon, France4Département de Biologie, Université de Fribourg, Fribourg, Switzerland

6.1 INTRODUCTION

The study of plant pathogens has been driven to a great extent by the problems they cause in thefield and was traditionally aimed at describing the micro-organisms, the infection process and

finding new approaches to contain them. More than 50 years ago, studies began to be aimed atthe host response to pathogens. Since then many researchers in search of fascinating biological

problems linked with potential practical applications have moved into the field of studyingplant-pathogen interactions. Nowadays, the resistance of plants to pathogens has become adynamic research topic that has witnessed great conceptual advances.

Plant defences comprise both pre-existing barriers as well as defences induced upon percep-tion of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) or molecules

produced from damage as a result of infection (DAMPs; damage-associated molecular pat-terns). Perception of such molecular patterns and the signalling thus initiated leads to the

deployment of a syndrome of induced defences (basal induced resistance), which includesthe amplification of the initial signals to neighbouring cells and even to other parts of the plant

systemic acquired resistance (SAR) or systemic induced resistance (ISR). This chapter willfocus mostly on the induced mechanisms of defence.

6.2 STRUCTURAL BARRIERS

The cell wall is considered to be the first obvious barrier to potential pathogens. Progress hasbeen made in elucidating its chemical structure and function (Burton et al., 2010). The cell

wall is also a source of molecules (DAMPs) that signal the presence of invading microbes

* These authors contributed equally to this chapter

Induced Resistance for Plant Defense: A Sustainable Approach to Crop Protection, Second Edition.Edited by Dale R. Walters, Adrian C. Newton and Gary D. Lyon.© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.


Mechanisms of Defence to Pathogens: Biochemistry and Physiology 107

and induce defence reactions (see reviews in Hückelhoven, 2007; Vorwerk et al., 2004; Wolf

et al., 2012). Many observations have shown that plants respond to attempted infections bythe formation of cell-wall deposits or papillae targeted at the site of attempted infection. Inmany cases, this provides an efficient barrier against non-host pathogens. True pathogens might

outrun the plant or prevent the deposition of papillae. A number of reviews have addressed thistopic in the past (Hückelhoven, 2007; McLusky et al., 1999; Nicholson and Hammerschmidt,1992, Thordal-Christensen, 2003; Thordal-Christensen et al., 1997; Zeyen et al., 2002).

6.2.1 Early events: The cytoskeleton and traffic of vesicles

Advances have been made in understanding the cellular organization leading to the formationof localized cell-wall appositions. Various studies have highlighted the contribution of the plantcytoskeleton, in particular the role played by actin filaments (Schmelzer, 2002). The reorgani-

zation of actin filaments in response to an attempted penetration by a microbe, as well as theaccumulation of associated proteins such as profilins and Rop GTPases, are among the firstresponses detected at an infection site (Day et al., 2011; Hardham et al., 2007; Hückelhoven,

2007; Lipka and Panstruga, 2005; Schutz et al., 2006). One current model holds that actinfilaments form a network targeted to the site of attempted penetration and presumably assistin directing proteins or vesicles containing various materials including cell-wall components

(typically see Shimada et al. (2006), reviewed in Hardham et al. (2007)).Studies on membrane traffic in relation to pathogen infection have intensified in recent years

(Lipka et al., 2007). The first studies supporting the importance of vesicle traffic focussed on

syntaxins, a group of proteins belonging to the t-SNARE family that mediate the fusion ofcargo vesicles to target membranes. For example, Arabidopsis is typically resistant to the bar-ley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) and this is associated with

secretion of cell-wall appositions to the penetration site. The importance of cell-wall depositsin this non-host interaction was demonstrated in mutants of the syntaxin AtSYP121/PEN1gene.The pen1 mutant shows increased penetration of Bgh together with a delay in the formation

of cell-wall deposits (Assaad et al., 2004; Collins et al., 2003). A similar observation was alsomade in barley (Collins et al., 2003). Arabidopsis also harbours AtSYP122, a close homologueof AtSYP121/PEN1, which additionally plays a part in secretion and cell wall deposition and

overlaps only in part with the function of SYP121 (Assaad et al., 2004). Only PEN1 is requiredfor targeted secretion that results in cell-wall deposits associated with resistance to Bgh. How-ever, AtSYP121 and AtsSYP122 negatively regulate defence reactions such as programmed

cell death, or defence signalling that is dependent on salicylic acid (SA), jasmonic acid (JA)or ethylene (ET) through a mechanism that is distinct from that deployed during resistance to

penetration (Zhang et al., 2007). The molecular basis for these opposing roles has yet to bestudied, but it is suggested that a cell will either display resistance to penetration or SA-, JA-,ET- and programmed cell death-mediated defences.

Further studies on the formation of cell-wall appositions during the defence response of bar-ley to powdery mildew have shown that ADP-ribosylation factor (ARF) GTPases are involvedin vesicle budding. Besides ARF-GTPases, mutant studies showed that a membrane-localized

syntaxin, known as REQUIRED FOR MLO-SPECIFIED RESISTANCE2 (ROR2), is alsorequired for resistance to penetration. Both ARDF and ROS2 function in the same vesiclepathway and ARF vesicular bodies accumulate at the penetration site before callose can be

detected in the papilla. The hypothesis was made that the callose might be conveyed to thepapillae at the site of penetration by means of ARF vesicles. So far, the production of callose


108 Induced Resistance for Plant Defense

was assumed to be at the site of cell-wall apposition (Bohlenius et al., 2010). New compo-

nents of vesicle complexes and of Golgi membrane trafficking involved in the resistance topenetration were recently identified in barley using an approach based on transient inducedgene silencing of candidate genes selected from protein databases (Ostertag et al., 2013). All

of these studies have shown that the cytoskeleton and the associated vesicles are instrumentalfor targeted deposition of cell wall appositions.

6.2.2 The nature of cell wall appositions

Papillae contain callose, a β-1,3-glucan polymer with β-1,6-glucan branches (Stone and

Clarke, 1992). Other components include various phenolics, hydrogen peroxide or proteins(Bestwick et al., 1997; Rey et al., 1996; Smart et al., 1986; von Röpenack et al., 1998; Zeyenet al., 2002). Callose is a major component of papillae and is produced after inoculation

with pathogens or with chemical potentiators of plant resistance (Hückelhoven et al., 1999;Skalamera and Heath, 1996; Soylu et al., 2004; Kogel et al., 1994; Ton et al., 2005; Zimmerliet al., 2000). During a resistant interaction, papillae enriched with callose might offer a

mechanical barrier to penetration by non-host pathogens, but virulent pathogens are notaffected either because they are faster or they can prevent its formation (Aist, 1976).

A number of reports have further explored the relevance of callose as a mechanical barrier

for penetration. In Arabidopsis Atgsl5, a gene encoding glucan synthase 5 catalyses callosebiosynthesis. This gene is highly expressed in constitutively resistant Arabidopsis mapk4(Ostergaard et al., 2002). Constitutive expression of the NahG gene encoding a bacterial SA

hydroxlyase in mapk4 abolishes resistance and expression of Atgsl5 (Ostergaard et al., 2002).The content of callose in papillae was reduced in double-stranded RNA interference linestargeted at Atgls5, but these plants only showed marginal loss of resistance to penetration

by a non-host powdery mildew from barley however displayed strong resistance againstPeronospora parasitica, the virulent powdery mildew of Arabidopsis (Jacobs et al., 2003).

Similarly, the powdery mildew resistant 4 mutants (pmr4) do not produce callose and

exhibit enhanced resistance rather than susceptibility to virulent powdery mildew pathogens(Nishimura et al., 2003). This paradoxal result was explained by a possible negative feedbackloop whereby callose limits excessive defences that might damage the cell (Nishimura et al.,2003). Another rationale was that the absence of callose at infection sites might expose andpromote pathogen-derived elicitors for defence, or that callose seals off the invader againstthe action of plant antimicrobials (Jacobs et al., 2003). Alternatively, the absence of callose

might prevent the formation of the neck septum at the base of the haustorium and nutrientsin the extrahaustorial space might diffuse into the apoplasm, thus impairing the survival of

the biotroph.The stress hormone abscisic acid (ABA) has been involved both as a positive (Asselbergh

and Höfte, 2007; Ton et al., 2009) and a negative regulator (Clay et al., 2009; de Torres-Zabala

et al., 2007) of callose formation. Environmental conditions (light, nutrients) strongly affectcallose formation and PAMP-induced callose formation in hydroponically grown Arabidopsis(Luna et al., 2010) from such model set-ups might therefore be misleading, as highlighted by

this study (Luna et al., 2010).The importance of callose deposition has recently been examined in engineered Arabidopsis

that constitutively overexpress the POWDERY MILDEW RESISTANT4 (PMR4) gene encoding

a callose synthase. The activity of the callose synthase was enhanced in the transgenic plantsand so was the size of the callose deposits. Interestingly, the constitutive deposition of callose



in the transgenic lines led to a strong resistance to penetration upon inoculation with either the

virulent powdery mildew Golovinomyces cichoracearum or with the non-pathogen Blumeriagraminis f. sp. hordei (Ellinger et al., 2013).

6.2.3 Lignification

The aromatic polymer lignin is a major component of secondary walls and has essential

functions in plant growth and development as well as in the defence against invaders. Lignin

makes the wall mechanically rigid and prevents diffusion of water-soluble compounds

(enzymes, toxins) released by pathogens (Ride, 1983). Its complex polymeric nature is slowly

being deciphered (Boerjan et al., 2003; Humphreys, 2002; Zhao and Dixon, 2011; Zubieta

et al., 2002).

Defence lignin refers to lignin deposited in response to pathogen invasion (Nicholson

and Hammerschmidt, 1992). Such defence lignin has been observed in cell-wall appositions

(Carver et al., 1992; von Röpenack et al., 1998) but also in entire walls of the infected

cells or only at the infection site (Heitefuss, 2001; Moersbacher and Mendgen, 2000; Vance

et al., 1980).

In addition, defence-related lignin might be of a different composition to developmentally

related lignin. As discussed by Hückelhoven (2007) the evidence for a role of lignin in resis-

tance is mostly based on correlative studies (with various inhibitors) and genetic evidence

using suppression of gene expression is rare, given the redundancy of the enzymes involved in

lignin biosynthesis. Despite this difficulty, Bhuiyan et al. (2009) recently silenced individually

or in combination several genes involved in monolignol synthesis in wheat using RNAi inter-

ference. The transcripts of phenylalanine ammonia-lyase, caffeic acid O-methyltransferase,

ferulic acid hydroxylase, caffeoyl-CoA O-methyltransferase and cinnamyl alcohol dehydro-

genase were found to accumulate differentially in the epidermis of susceptible or resistant

plants after infection with Blumeria graminis f. sp. tritici (Bgt). The transient silencing of these

genes in this cell layer led to an increased susceptibility to Bgt and decreased resistance to pen-

etration against the non-host pathogen Blumeria graminis f.sp. hordei. The autofluorescence

of the papillae at the site of contact with the pathogen was also decreased, providing evi-

dence for a role of monolignol production in localized defence to pathogens in wheat (Bhuiyan

et al., 2009).

6.3 PHYTOALEXINS

6.3.1 The concept of phytoalexins

The first experimental evidence for the occurrence of antibiotic plant metabolites induced by

pathogen challenge was provided by Bernard (1911; cited in Grayer and Kokubun (2001))

and Müller and Börger (1940). This led to the concept of phytoalexins (from Greek alexein,

to defend) defined as ‘low molecular weight, antimicrobial compounds that are both synthe-

sized by and accumulated in plants after exposure to microorganisms’ (Paxton, 1981). They

are distinguished from phytoanticipins, which refer to ‘low molecular weight, antimicrobial

compounds that are present in plants before challenge by micro-organisms or are produced

after infection solely from pre-existing constituents’ (VanEtten et al., 1994). Both classes of

molecules have classically included secondary metabolites and not antimicrobial peptides.



We will keep this distinction in this section and focus on phytoalexins, but many of the concepts

reviewed here can be applied to phytoanticipins.

The inducibility of phytoalexin biosynthesis has probably been favoured in the course of

evolution by biological constraints such as metabolic costs and functional side-effects associ-

ated with chemical defence (but see Neilson et al., 2013).

6.3.2 Distribution of phytoalexins among taxonsand individuals

In a review published in 1999, Harborne indicated that more than 300 molecules had been

identified as phytoalexins from approximately 900 species representing 40 plant families

(Harborne, 1999). These numbers must have increased since then, as new phytoalexins have

been discovered, for instance in peanut (Sobolev et al., 2009; Sobolev et al., 2011), maize

(Huffaker et al., 2011; Schmelz et al., 2011), wheat (Du Fall and Solomon, 2013) and rice

(Inoue et al., 2013). All of these compounds can be grouped into structural families and

related by their biosynthetic pathways. A close association exists between some structures

and taxa, for example isoflavonoids are mainly produced by the Papilionoideae subfamily

of Leguminosae, sesquiterpenes by Solanaceae, sulfur-containing indoles by Brassicaceae

(Harborne, 1999; Grayer and Kokubun, 2001). On the other hand, some phytoalexins are

shared by widely divergent plant species, such as stilbenes that occur in peanut, grapevine and

pine. A single species may produce several related and unrelated phytoalexins; for instance,

in rice, 16 different phytoalexins have been isolated, although it is not known if all of these

compounds are relevant for defence. Leaves and roots of Arabidopsis do not produce the

same antimicrobials (Bednarek et al., 2005).

6.3.3 Biosynthetic pathways and their regulation

The number of major biosynthetic pathways is small relative to the wide chemical diversity

of phytoalexins, if one excludes nitrogen-containing phytoalexins. This provides a simple

way to organize and classify these compounds (Figure 6.1). Combinations of pathways and

subsequent modifications (hydroxylations, methylations, cyclizations, etc.) generate extensive

divergence within each structural family. Many phytoalexins belong to the phenylpropanoid

family, characterized by the C6C3 skeleton of phenylalanine. The entry point into this class

of molecules is catalyzed by phenylalanine ammonia lyase (PAL) through the deamination of

phenylalanine into trans-cinnamic acid. Some phytoalexins are readily formed from this com-

pound, for example p-coumarate (Daayf et al., 1997), or from dimerization and further modi-

fications of relative compounds, for instance lignans, a representative of which is matairesinol

(Lewis and Davin, 1999; Umezawa, 2003).

Trans-cinnamate and related molecules can also undergo cyclization, giving rise to a

coumarin skeleton, for example scopoletin and umbelliferone (Matern et al., 1999), which

can in turn be prenylated, producing furano- and pyrano-coumarins, for instance xanthotoxin

(Stanjek et al., 1999; Larbat et al., 2009). The C6C3 skeleton can also be extended by

the activity of polykeptide synthase (PKS) enzymes (see review by Flores-Sanchez and

Verpoorte, 2009), as exemplified by chalcone synthase (CHS) and stilbene synthase (STS)

that generate committed precursors of the flavonoid and stilbenoid families, respectively.

Flavanones can be further processed into isoflavonoids, through activity of isoflavonoid

synthase, and subsequently modified by numerous enzymes (Dixon et al., 1995; Wang, 2011)



3 × C2 unitsOPP

OH

O

O

CHO

HO

OPP

OH

O

O

HO

HO

COOH

CH2OH

O

H

H

O

NH2

OH

HO

HO OH

OOH

HO O OMeO

O

OH

O

O

O O

O

O O

O

OH

OH

O

O

O

HO

OH

OH

OH

OH

OH

OH

OH

HO O+

O

OMeO

OH

O

O

COOMe

MeO

OH O

OO

MeO

HO

O

O

OMe

OH

HO O O

O

SCoA

OO

OMe

O OH

OMe

OMe

IPP

trans-cinnamic acid

Phenylalanine

DMAPP

2,7-DihydroxycadaleneLettucenin A

Momilactone A

Arjunolic acid

Chalcone(−)-Matairesinol

6-Methoxymellein

Wyerone

Sakuranetin

Phaseollin

Glyceollin I

Resveratrol

Luteolidin(+)-Pisatin(+)-Maackiain

PAL

CHS

STS

oleate

Lignanes

Flavanones

Stilbenoids

3-deoxyanthocyanidins

Sesquiterpenes (C15)

Diterpenes (C20)

Triterpenes (C30)

Isoflavonoids

Mevalonate 1-Deoxyxylulose

DMAPP

Umbelliferone

Xanthotoxin

Coumarins

DMAPP

TERPENOIDS

PHENYLPROPANOIDS

POLYKETIDES

FNRIFS

Aucuparin

Biphenyls

benzoyl-CoA

BIS

3 × C2 units

3 × C2 units

Fig. 6.1 Overview of the elaboration of the carbon skeleton of terpenoid, phenylpropanoid and polyketide phytoalexins. Only selected examples are shown.Phytoalexin names are in italic, enzymes in bold, generic classes of compounds are underlined. Plain arrows indicate a reaction in a single step, dashed arrowsrepresent several consecutive enzymatic steps. Abbreviations: BIS, biphenyl synthase; CHS, chalcone synthase; DMAPP, dimethylallyl pyrophosphate; FNR,flavanone reductase; IFS, isoflavone synthase; IPP, isopentenyl pyrophosphate; PAL, phenylalanine ammonia lyase; and STS, stilbene synthase.



producing pisatin, phaseollin or glyceollin, for example. In sorghum, apigeninidin and

luteolinidin 3-deoxyanthocyanidin phytoalexins also stem from flavanones following catal-

ysis by flavanone-4-reductase (FNR) (Liu et al., 2010). The resveratrol produced by STS is

a phytoalexin on its own but also constitutes a precursor of other antimicrobial compounds

(Jeandet et al., 2010). Biphenyls and dibenzofurans, for instance aucuparin, are generated

from benzoyl-CoA through the activity of biphenyl synthase (BIS), another PKS enzyme

(Chizzali and Beerhues, 2012). Benzoyl-CoA acid itself is produced from trans-cinnamate

(Gaid et al., 2012; Klempien et al., 2012; Qualley et al., 2012).

Besides phenylpropanoids, terpenoids also form a structural family encompassing many

phytoalexins. The precursors isopentenyldiphosphate (IPP) and dimethylallyldiphosphate

(DMAPP) are generated through the cytosolic mevalonate pathway or through the plastidic,

1-deoxyxylulose pathway (Hemmerlin et al., 2012). Assembly of the C5 chain of IPP and

DMAPP, and of the resulting products by terpene synthases, yields isoprenoids of several

carbon chain lengths (C10, C15, C20 or C30) that are further modified by specialized enzymes

(Liang et al., 2002). Some examples of terpenoid phytoalexins include 2,7-dihydroxycadalene,

momilactone A or arjunolic acid. A few phytoalexins also rely on condensation of acetate

units, after previous activation in the form of malonate, for the elaboration of their carbon

skeleton. Certain PKS enzymes, belonging to the same superfamily as CHS and STS, mediate

these reactions. For instance, wyerone arises from desaturation and cyclization of its precursor

oleate, produced by the PKS fatty acid synthase (Nawar and Kuti, 2003). Another example

is 6-methoxymellein, whose precursor 6-hydroxymellein is generated by a dedicated PKS

(Kurosaki, 1994; Fan et al., 2000).

Certain phytoalexins, in particular those containing nitrogen, are produced by yet more

different pathways, as shown by the indole-based phytoalexins of Brassicaceae (Pedras et al.,2011). Recent developments on camalexin biosynthesis in Arabidopsis have been reviewed in

Ahuja et al. (2012).

Following biotic or abiotic elicitation, dedicated transcription factors were found to

coordinate the expression of whole phytoalexin biosynthesis pathways through transcriptional

control (Dixon and Paiva, 1995; Dixon et al., 1995; Zhao et al., 2005). In sorghum, the

MYB transcription factor YELLOW SEED1 has been associated with accumulation of three

deoxyanthocyanidin phytoalexins (Ibraheem et al., 2010). In rice, the basic leucine zipper

OsTGAP1 transcription factor is sufficient to induce the production of the momilactones

and phytocassane phytoalexins (Okada, 2011). In Arabidopsis, the WRKY33 and other

unidentified WRKY transcription factors target and activate several genes of the camalexin

biosynthesis pathway (Ahuja et al., 2012; Birkenbihl et al., 2012). These examples of

transcriptional activation are the end-result of signalling processes that can involve key

signalling molecules such as SA, JA and other prominent players in defence signalling. The

induction of phytoalexin production upon elicitation or pathogen challenge has been reviewed

recently for Arabidopsis and crop plants of the Fabaceae, Solanaceae, Vitaceae and Poaceae

families (Ahuja et al., 2012).

Further elements may contribute to the regulation of phytoalexins biosynthesis. In

the genome of rice, the genes involved in momilactone and phytoacassane biosynthesis

are organized into two clusters (Yamane, 2013). The biological significance of such an

arrangement remains intriguing (Field et al., 2011). Metabolic channelling could also act as a

post-translational regulation. In this process, successive enzymes of a pathway are associated

by specific interactions, allowing for rapid channelling of the substrate from one active



site to another and avoiding loss or dilution into the intracellular compartment (Sweetlove

and Fernie, 2013). This metabolic efficiency could be one of the keys to the rapid productionof the high amounts required for phytoalexin efficiency. So far, metabolic channelling hasbeen shown to be effective in the isoprenoid, PAL and flavonoid pathways (Jørgensen et al.,2005; Neilson et al., 2013, Winkel, 2004).

6.3.4 Role of the phytoalexins in the defence response

Induced accumulation of a metabolite following pathogen infection might suggest a functionfor this molecule in plant defence, but nevertheless a complete demonstration would require

further investigations. The criteria to examine the relevance of a phytoalexin as a defencemechanism during the plant pathogen interaction include: (1) the compound must accumulatein response to infection; (2) the compound must be inhibitory to the invading pathogen; (3) the

compound must accumulate up to inhibitory concentrations in the vicinity of the pathogen atthe time it ceases growing in the plant; (4) variation in the rate of accumulation of the phy-toalexin should cause a corresponding variation in the resistance of the plant; and (5) variation

in the sensitivity of the invading organism should cause a corresponding variation in its vir-ulence. With the exception of point (1), these criteria were originally postulated to examinethe importance of phytoanticipins (Wood, 1967). The various lines of evidence showing the

involvement of phytoalexins in plant defence will be reviewed subsequently, using the frame-work of the criteria mentioned earlier in this paragraph.

Phytoalexins are toxic towards a wide range of organisms, including bacteria, fungi, nema-

todes and higher animals, and even plants themselves. The EC50 (effective concentration forproducing 50% of inhibition) for fungi usually ranges from 10−3 to 10−5 M, and the MIC (min-imum inhibitory concentration) for bacteria lies between 100 and 1000 μg ml−1, classifying

phytoalexins as relatively weak antifungal and antibacterial agents (Kuc, 1995; Tegos et al.,2002) thus raising the issue of their actual concentration in the close vicinity of the pathogen.A number of studies have documented phytoalexin production at the site of pathogen attack

(Cooper et al., 1996; Hahn et al., 1985; Kliebenstein et al., 2005; Schuhegger et al., 2007;Simon et al., 2010; Snyder and Nicholson, 1990; Sobolev, 2008; Yoshikawa et al., 1978).Moreover, the synergy between the various compounds secreted by plants probably greatly

increases their antimicrobial efficiency (Lewis and Ausubel, 2006). Last, but not least, apartfrom their direct antimicrobial activity, some phytoalexins might also play a role in defencethrough signalling or other mechanisms (Bednarek, 2012; Bednarek and Osbourn, 2009).

Many studies have established a correlation between phytoalexin accumulation and resis-tance to disease, although correlative evidence has to be further tested (Kuc, 1995). One of the

best pieces of evidence available was provided by the transfer into different host plants of thestilbene synthase gene catalysing the one-step formation of resveratrol from the two ubiqui-tous plant metabolites p-coumarate and malonate (Figure 6.1). Introduction of this gene results

in increased resistance of tobacco to Botrytis cinerea (Hain et al., 1993), and of many cropplants against different pathogens (Zhu et al., 2004 and references cited therein), although insome specific pathosystems, no effect was observed (Giorcelli et al., 2004; Kobayashi et al.,2000). Constitutive expression of isoflavone O-methyltransferase, catalysing a key reactionin flavonoid biosynthesis, increased resistance of alfalfa to Phoma medicaginis, even if theendogenous gene was induced after infection (He and Dixon, 2000). Conversely, mutants

or transgenic plants specifically affected in phytoalexin biosynthesis are more susceptiblethan the corresponding wild types. The pad3 mutant of Arabidopsis is defective in camalexin



biosynthesis and exhibits a greater susceptibility than the parental line to several, but not all,

pathogens (Ahuja et al., 2012; Kliebenstein, 2004). Inhibition of the chalcone synthase in

cucumber and silencing of isoflavone synthase or chalcone reductase genes in soybean lead

to enhanced susceptibility to diseases, confirming that induced resistance in these species is

linked to flavonoid phytoalexin accumulation (Fofana et al., 2005; Graham et al., 2007; Subra-

manian et al., 2005). Similarly, engineered pea plants with reduced rates of pisatin production

were more susceptible than the wild-type controls (Wu and VanEtten, 2004). These examples

are also discussed in more detail in a recent review (Jeandet et al., 2013).

Virulent pathogens were generally found to be more tolerant to phytoalexins of their host

than avirulent or non-pathogenic organisms (VanEtten et al., 2001). An excellent and thor-

oughly studied example is the degradation of the pea phytoalexin pisatin by virulent strains of

Nectria haematococca (member of the Fusarium solani species complex). Some strains of this

fungus possess a PDA gene encoding a cytochrome P450 enzyme that demethylates pisatin into

a less toxic compound (VanEtten et al., 2001). Gain- and loss-of-function experiments with the

PDA gene demonstrated that pisatin detoxification is an important component of pathogenicity

on pea in N. haematococca (Ciuffetti and VanEtten, 1996; Wasmann and VanEtten, 1996) or

in pathogens normally not pathogenic on pea (Schäfer et al., 1989). Transcription of the PDA

gene is induced by pisatin, revealing that the presence of pisatin can trigger signalling in the

pathogen (Khan et al., 2003). The detoxification of host phytoalexins through various enzy-

matic modifications has been reported for many virulent pathogens (reviewed by Pedras and

Ahiahonu, 2005). Although the PDA gene was acquired by horizontal gene transfer between

closely related pea pathogens, simultaneously to other virulence genes (Milani et al., 2012), the

data accumulated so far suggest that pathogens can evolve independent and specific solutions

to phytoalexin detoxification (Pedras and Ahiahonu, 2005; Pedras et al., 2011).

Other mechanisms can account for the tolerance of bacteria and fungi to toxic compounds

produced by plants (VanEtten et al., 2001). N. haematococca also circumvents the toxicity

of pisatin by a non-degradative mechanism based on an efflux pump (Coleman et al., 2011).

Experimental challenges with mutants of such efflux transporters indicated that these extru-

sion systems are an important factor of virulence, as shown not only for N. haematococca(Coleman et al., 2011), but also for other fungal and bacterial pathogens (Barabote et al.,2003; Burse et al., 2004; Fleissner et al., 2002; Schoonbeek et al., 2001; Stefanato et al.,2009; Stoitsova et al., 2008; Urban et al., 1999; Vargas et al., 2011). It was proposed that in N.haematococca, the energy consumption of the pisatin efflux transporter activates the pisatin

degradation activity (Coleman et al., 2011). In Pseudomonas syringae pv. tomato, various

flavonoids directly induce conformational changes of the PmeR transcriptional repressor, lead-

ing to an increased expression of the MexAB-OprM multidrug extrusion system (Vargas et al.,2011). In the never-ending warfare between plants and pathogens, countermeasures against

phytoalexin efflux pumps presumably appeared, and exciting reports are now describing the

isolation from plant tissues of inhibitors of multidrug extrusion systems, although not yet in the

context of plant–pathogen interactions (Belofsky et al., 2004; Ettefagh et al., 2011; Fiamegos

et al., 2011; Kalia et al., 2012; Morel et al., 2003; Reimann and Deising, 2005; Stermitz et al.,2000). Such inhibitors are expected to dramatically increase the sensitivity of several bacterial

plant pathogens to plant antimicrobial metabolites (Tegos et al., 2002), and are of considerable

interest for medical applications (González-Lamothe et al., 2009; Lewis and Ausubel, 2006).

Finally, another possible strategy used by pathogens to avoid phytoalexin toxicity is to block

or reduce their production using effectors. The HopZ1 effector of Pseudomonas syringae



binds to and reduces levels of the soybean 2-hydroxyisoflavanone dehydratase, required forisoflavone biosynthesis, and decreases the amount of daidzein produced after infection ofsoybean (Zhou et al., 2011). Even though this work did not establish the exact role of theisoflavonoid phytoalexins in this pathosystem (see comment by Bent, 2011), it shows thatphytoalexin production can be altered by pathogens through the use of effectors.

6.4 THE HYPERSENSITIVE RESPONSE (HR)

6.4.1 In the death car – en route to plant resistanceto pathogens

Historically, the term ‘hypersensitive’ refers to the rapid and localized cell death induced inspecific cereal cultivars by the fungal pathogen Puccinia graminis (Stackman, 1915). Theexpression ‘hypersensitive response’ (HR) was coined later on when it appeared that this formof plant cell death was generally associated with resistance to many pathogens (Goodman andNovacky, 1994). From accumulating data, however, it now seems clear that pathogen-triggeredcell death can be dissociated from defence mechanisms and, to some extent, from plant resis-tance (Clough et al., 2000; Coll et al., 2010; Yu et al., 1998). That is why the term HR iscommonly used in the literature for describing the defensive arsenal that is deployed duringincompatible interaction and plant host cell suicide is often referred to as hypersensitive celldeath (HCD) in this context.

HCD is a form of programmed cell death (PCD), which implies a genetic orchestrationof cell suicide. HCD can be induced following plant challenge with viruses, bacteria, fungi ornematodes. It is characterized by the rapid collapse of tissues at the attempted site of infection,leading to the formation of highly localized lesions. Although it may currently be a mat-ter of debate for experts, it is generally assumed that HCD contributes to plant resistance tobiotrophic pathogens by preventing their feeding as well as spreading into healthy adjacent tis-sues. Thus, in line with the guard hypothesis (Dangl and Jones, 2001; Jones and Dangl, 2006),intrusion of avirulent pathogens is directly or indirectly perceived by plants through molecu-lar perturbations they occasion within host cells. Subsequently, when HR is launched it mostoften culminates in HCD. By contrast, virulent pathogens can escape this surveillance sys-tem and, remaining incognito in planta, they can interfere with defence onset and inhibit HR,including HCD. This results in intruder propagation and disease development. Interestingly,the pathogen lifestyle may also dictate the outcome of HR. When dealing with necrotrophicinvaders for instance, HCD execution may be detrimental to plants since these microorganismsfeed and live on dead tissues (Morel and Dangl, 1997).

In plants, PCD processes cannot readily fall into the comfortable three classes defined bythe morphological, molecular and biochemical criteria used for animal models, that is, apop-totic, autophagic and lysosomal cell death (Schweichel and Merker, 1973). In addition, HCD,which is an active phenomenon, is barely classable (van Doorn et al., 2011). Depending onpathosystems, it can either exhibit partial apoptotic or full autophagic morphotypes, or evendisplay unusual morphotypes reflecting plant-specific modes of cell-death orchestration (asdiscussed later). On the one hand, this strongly suggests that HCD and some animal cell deathforms can share cogs of their execution/regulatory machineries. This hypothesis is substanti-ated by the functional conservation across kingdoms of autophagy as well as a few apoptosisregulators, such as BAX INHIBITOR-1 and DEFENDER AGAINST APOPTOTIC CELL



DEATH-1 (Chae et al., 2003; Danon et al., 2004; Hückelhoven, 2004). On the other hand, this

further points out the selection of plant-specific death executioners through the course of evo-

lution for successful development of HCD. For instance, fatty acid hydroperoxides produced

by lipoxygenases could represent some of these executioners. Hence, while the occurrence of

multiple cell-death pathways may confer evolutionary advantages to plants facing biotrophic

pathogen attacks, it also raises the fascinating question as to how cells engage in one pathway

rather than another one. In other words, in which way the death car is regulated?

6.4.2 The role of reactive oxygen and nitrogen species(ROS and RNS)

Since the first report indicating that O2⋅− production is induced in potato during its interaction

with Phytophthora infestans (Doke, 1983), several plant tissues and suspension-cultured cells

have been reported to produce ROS after pathogen infection. ROS are chemically reactive

species of oxygen formed by successive one-electron reduction of molecular oxygen (O2)

and include the superoxide anion (O2⋅−), hydrogen peroxide (H2O2), hydroxyl radical (OH⋅)

or hydroperoxyl radical (HO2⋅). ROS are also generated during plant development and by a

plethora of environmental factors (Laloi et al., 2004).

After pathogen infection or elicitor treatment, the most abundant ROS produced is H2O2

and its production is mainly observed in the apoplastic space and coincides with the induction

of cell death during the HR (Grant and Loake, 2000 and references cited within). Modulation

of ROS levels in planta by lowering catalase or ascorbate peroxidase activity has demonstrated

the role of H2O2 in limiting pathogen spread and suggested its involvement in cell death (Dat

et al., 2003; Mittler et al., 1999). The use of either pharmacological or genetic approaches

(O’Brien et al., 2012a) has shown that, during plant defence, ROS production comes either

from the dismutation of O2⋅− generated by membrane-bound respiratory burst oxidase homo-

logue (RBOH) proteins, which are the orthologues of the gp91phox subunit of the macrophage

NADPH oxidase (Torres et al., 2002), or by the cell wall bound class III peroxidases that can

directly produce H2O2 under specific conditions and the provision of a strong reductant (Bind-

schedler et al., 2006; O’Brien et al., 2012a). Recently, the mitochondria as a source of ROS

during plant–pathogen interaction has been described (Gleason et al., 2011).

The mutation or the silencing of the genes encoding RBOH proteins has been shown to

correlate with a reduced HR in different species. For instance, single and double mutants of

Arabidopsis RbohD and RbohF genes display reduced ROS production and a reduced HR after

inoculation with avirulent Pseudomonas syringae pv. tomato bacteria (Torres et al., 2002).

Similar observations have been made in Nicotiana benthamiana. Silencing of both NbRbohA and B results in a reduced production of H2O2, a delayed and a reduced HR following the

infiltration of Phytophthora infestans elicitor INF1. However, Zhang et al. (2009) showed that

silencing of both proteins did not impact the HR induced by various elicitors, including INF1

or harpin, but is rather involved in the stomatal closure induced by these elicitors.

Although RBOH D and F proteins are crucial to the generation of ROS during plant

defence in Arabidopsis, their role in mediating disease resistance to microbial pathogens

is not clearly established. Whereas they do not seem to be important for the resistance to

avirulent Pseudomomas syringae pv. tomato bacteria carrying the Rpm1 gene (Chaouch et al.,2012; Torres et al., 2002) other studies have reported their partial importance in response to

virulent Pseudomonas bacteria (Chaouch et al., 2012; Daudi et al., 2012; Torres et al., 2002).



Silencing of NbRboh A and B in N. benthamiana has also resulted in a greater susceptibility

of the plants to the infection by Phytophthora (Yoshioka et al., 2003).

Haeme-containing cell wall bound class III peroxidases are also able to produce ROS in

various species (O’Brien et al., 2012a). In French bean, ROS production is at least generated

by the cell-wall peroxidase FBP1 (French Bean Peroxidase 1; Blee et al., 2001). By the overex-

pression of an antisense cDNA of FBP1 in Arabidopsis, Bindschedler et al. (2006) have shown

that these plants exhibit a strongly reduced ROS production and a hypersusceptibility to vir-

ulent and avirulent strains of Pseudomonas syringae and fungal pathogens including Botrytiscinerea. This strategy has led to the identification of AtPRX33 and AtPRX34, two FBP1

orthologues in Arabidopsis. Mutations of both protein-coding genes have indicated that the

peroxidase-dependant ROS production may play a role in MAMP-elicited defence responses

and more generally in plant defence to pathogen infections (Daudi et al., 2012; O’Brien et al.,2012b). However, the phenotype of atprx33/34 mutants are not as strong as the one of FBP1

antisense lines, suggesting that other peroxidases could be involved in this process. However,

the role of ROS-peroxidase generation in mediating HR has not yet been assessed.

Regarding the role of ROS during pathogens attack, some experiments have also indicated

that ROS inhibit spore germination of some pathogens in vitro suggesting a similar role invivo during infection (Peng and Kuc, 1992). However, it is now clear that the oxidative burst

observed in response to pathogens or other stimuli is not only deleterious for cells (see later)

or pathogens but participates in cellular signalling leading to plant defence. A pool of genes

whose expression depends on ROS production has been identified by microarray profiling

(Desikan et al., 2001; Gechev et al., 2005) and ROS have been implicated in cellular signalling

associated with the induction of defence processes (reviewed by O’Brien et al., 2012a; Petrov

and Van Breusegem, 2012).

NO serves as a signalling molecule in plants as it does in animals (Besson-Bard et al.,2008). Its role during HR is now established. Cell death induced by exogenous NO treatment

exhibits morphological features observed during plant PCD (Clarke et al., 2000; Neill et al.,2003; Zottini et al., 2002).

Pharmacological experiments using mammalian NOS (nitric oxide synthase) inhibitors or

NO scavengers have indicated the role of NO in triggering plant defence and plant cell death

in different models (Bellin et al., 2013). For instance, animal NOS inhibitors reduce the extent

of HR induced by avirulent P. syringae in Arabidopsis, and this is accompanied by more

extensive bacterial growth in treated tissue, suggesting that HR prevents pathogen spreading

through an NO-dependent pathway (Delledonne et al., 1998). In agreement with these obser-

vations, plants expressing the NO-scavenger haemoglobin express a reduced HR in response to

Tobacco necrosis virus and to avirulent P. syringae (Seregélyes et al., 2004). Scavenging NO or

inhibiting NO synthesis also delayed the cell death in elicitor-treated tobacco cell suspensions

(Lamotte et al., 2004).

Nevertheless, the role of NO during HR is not fully understood. Peroxynitrite (ONOO−)

formed by the reaction between NO and O2⋅−, has been detected during plant defence (Gaupels

et al., 2011). Peroxynitrite is highly toxic to mammalian cells where it accounts for most of

the cytotoxicity attributed to NO (Vandelle and Delledonne, 2011). In plants, treatment with

a high concentration of peroxynitrite or peroxynitrite donors failed to induce cell death in

some studies but not in others (Alamillo and García-Olmedo, 2001; Delledonne et al., 2001;

Romero-Puertas et al., 2007). Alamillo and Garcia-Olmedo (2001) also suggested a critical

role of peroxynitrite in triggering HR since its scavenging by urate inhibits the HR induced by



avirulent P. syringae. However, others have shown it is a cooperation between NO and H2O2,produced from the dismutation of superoxide anions, that is needed for the triggering of celldeath rather than the accumulation of peroxynitrite (Delledonne et al., 2001; de Pinto et al.,2006, de Pinto et al., 2002). Furthermore, urate does not inhibit cell death triggered by the elic-itor cryptogein that also induce a concomitant ROS and NO production (Lamotte et al., 2004).

Recently, it has been shown that NO directly modulates the activity of the ROSproducing-enzyme AtRBOHD by S-nitrosylation (Yun et al., 2011). AtRBOHD isS-nitrosylated at Cys890 during infection by avirulent P. syringae bacteria. They showedthat AtRBOHD S-nitrosylation decreases its NADPH oxidase activity probably by impedingcofactor binding (FAD) and then reduces ROS accumulation, limiting cell death duringthe HR. NO can also impact mitochondrial functionality by the inhibition of cytochrome coxidase leading to a reduction of global cell respiration that partially mediates cell death(Millar and Day, 1996; Zottini et al., 2002).

Finally, experimental data indicate that NO impacts cell death through its signalling prop-erties rather than through direct toxic effects, even though such a deleterious role could not beexcluded.

6.4.3 On the highway of hypersensitive cell death: Signallingand regulation

En route to HCD, plant cells can take many distinct itineraries to reach the same final desti-nation, including bus itineraries in the case of traffic jams or because of roads under recon-struction. Thinking of this as a giant traffic map, it is therefore easy to imagine the complexityof the tangled hypersensitive signalling networks, most likely decorated by regulatory nodesresembling tolls. In addition, at the starting point of the road network, right after pathogen per-ception, there are many parallel ways leading, for instance, to reactive oxygen species and/orreactive nitric oxide species synthesis (discussed above), but they lack traffic signs so that it isnot obvious whether they end up in an HCD via the defence stop or they go straight to eitherone of the two destinations. Nonetheless, despite these difficulties, a few parts of the HCDhighway-forming web currently emerge from the fog due to efforts in the scientific community,pinpointing multilayered regulation levels.

In the early 1980s, the isolation of maize mutants showing spontaneous lesions undernon-permissive conditions corroborated numerous quantitative genetic studies, suggesting thatHCD is genetically programmed (Hoisington et al., 1982). Since then, these types of mutants,so-called lesion-mimic mutants (LMM), have been identified in additional plant species suchas rice and Arabidopsis (Kurata et al., 2005; Lorrain et al., 2003). One such approach hasproven instrumental for elucidating the signalling and regulation networks underpinning thedemise of hypersensitive cells. Not only did it allow for the identification of key cell deathmediators, but it also revealed unexpected regulatory mechanisms at the cell, tissue and evenorganism levels. Indeed, in the absence of pathogens, initiation LMM can exhibit localizednecrotic spots, whereas propagation LMM are unable to control the extent of hypersensitivelesions once initiated, a phenomenon known as runaway cell death (RCD). These data clearlyindicate that plants possess the molecular machinery to trigger HCD at will and efficientlyrestrict its spreading in healthy tissues upon pathogen attack.

During the last decade, major breakthroughs in our understanding of RCD have beenachieved. Performing a virus induced gene silencing (VIGS) based high-throughput screenin an attempt to identify tobacco HCD regulators, Liu et al. (2005) isolated ATG6/BECLIN1



as a suppressor of TMV-induced cell death. ATG6/BECLIN1 encodes a protein involved in

macro autophagy (previously and hereafter referred to as autophagy), more specifically in the

nucleation of autophagosome vesicles. ATG6-deficient lines displayed a typical RCD that

eventually engulfed the whole plant while viruses were not found outside of the inoculated

area. Silencing other autophagy-related genes also led to the observation of a comparable

phenotype following viral infection, pointing to autophagy as part of the machinery dedicated

to the restriction of HCD development in response to TMV.

Testing for additional pathosystems in tobacco (bacteria, oomycete elicitors, fungal

and bacterial effectors) and Arabidopsis plants (Patel and Dinesh-Kumar, 2008) further

suggested that the cytoprotective function of autophagy during HCD may be a conserved

feature. Remarkably, Bourque et al. (2011) also reported on cell death misregulation in

cryptogein-elicited tobacco plants affected in type-2 histone deacetylase. Loss-of-function

strategy combined with a pharmacological approach revealed exacerbated HCD at the

infiltrated foliar area associated with hypersensitive symptoms in untreated distal tissues.

Beyond the interesting potential relationship between autophagy and epigenetic phenomena

that awaits further investigations, these two works, supported by others (Coll et al., 2010;

Cui et al., 2013), underline the possibility that yet-uncharacterized pro-death signals could be

generated in infected dying zones and move into healthy systemic organs. Based on the work

of Liu et al. (2005), it is also tempting to speculate that pro-death signals are literally consumed

by autophagosomes in wild-type plants in order to neutralize them and prevent the onset of

undesired systemic HCD.

While refining the landscape around the autophagy toll, more itineraries and tolls are

progressively moving out of the fog. For instance, two Arabidopsis distant relatives of

animal caspases, metacaspases 1 and 2 (AtMC1 and AtMC2), were recently found to control

bacteria-induced HCD (Coll et al., 2010). AtMC1 positively regulates RPM1-coordinated cell

death, probably through its interaction with the protein LESION-SIMULATING DISEASE 1

(LSD1), whereas AtMC2 antagonizes this effect independently of its catalytic activity. Based

on previous work (Mühlenbock et al., 2008) showing a relationship between LSD1-dependent

immunity and light acclimation, these data link both AtMC1 and AtMC2 to chloroplasts,

revealing a new toll on the HCD highway. Interestingly, the outstanding work of Blanvillain

et al. (2011), which brought to light a small peptide named KISS-OF-DEATH as a potentially

novel mediator of HCD, further supported the case for mitochondria as another regulatory

node on our traffic map. However, at this stage of our knowledge, it still remains difficult

to connect all these tolls (chloroplasts, mitochondria and others) together in order to draw

complete itineraries.

This fact having being established, yet another new trend in the field strongly suggests a

mandatory visit to a geographical place, towards which many, if not all, roads would converge:

this is the endoplasmic reticulum (ER). Besides being invoked in synthesis, modification and

maturation of most secreted proteins, including defence proteins, the ER recently appeared

as an ubiquitous biotic/abiotic stress-sensing organelle in plants (Cacas, 2010). A growing

body of findings indicates that microbe effectors can target retrograde ER-to-nucleus signalling

pathways (Bosis et al., 2011; McLellan et al., 2013; Qiang et al., 2012) most likely for hijack-

ing the host defence response and manipulating HCD. Moreover, it has been also demonstrated

that ER homeostasis is of utmost importance for controlling HCD onset in tobacco plants chal-

lenged with bacterial, fungal and viral effectors (Xu et al., 2012). Therefore, it seems that future



challenges in the field would consist of ascertaining axes, connecting roads and placing tollsin order to build this giant signalling highway network that will ultimately lead to HCD.

6.4.4 License to kill: Where do we stand on executionof hypersensitive cell death?

Beyond the theoretical point of no return, which could correspond to the last exit(s) of adead-end highway, cells have no choice and must engage in the execution phase of HCD, dur-ing which they organize their self-dismantling. However, which exit should be taken? Animalcells possess a wide arsenal of well-documented death effectors destined towards their owndemise, such as caspases and autophagosomes. This is the same for plant cells, which haveevolved additional specific mechanisms and executioners – even though such roads/pathwaysare still under characterization.

As mentioned earlier, autophagy can exert a pro-survival function in plants, prevent-ing uncontrolled propagation of HCD (Liu et al., 2005; Patel and Dinesh-Kumar, 2008).Recently, Hofius et al. (2009) analysed HCD intensity in relation to autophagic response inArabidopsis upon inoculation of distinct avirulent P. syringae strains (DC3000::AvrRpt2;DC3000::AvrRps4 and DC3000::AvrRpm1). The first strain did not activate any autophagicvesiculation in cytoplasm of WT cells, indicating that execution of RPS2-dependentcell death is independent of autophagy. By contrast, the two other strains did trigger anautophagosome-contingent process in WT plants and autophagy-deficient mutants showedunexpected behaviours. On the one hand, knocking out ATG genes was responsible for asignificant delay in DC3000::AvrRps4-initiated cell death, strongly suggesting a pro-deathfunction for autophagy under these specific conditions. On the other hand, a marked decreasein hypersensitive lesion rate was only observed when atg mutants were coinfiltrated with bothDC3000::AvrRpm1 and cathepsin B inhibitors. In addition to a death-promoting functionfor autophagy, the latter data further point out the possibility that multiple execution deathpathways could cooperate simultaneously in the same plant cell.

With regards to the animal literature, the antagonistic outcomes of autophagy in plantimmunity might not be surprising since this process can either protect mammalian cells fromapoptosis or be used as a tool destined for cellular dismantling (Berry and Baehrecke, 2007;Samara et al., 2007). However, it still remains to be determined whether pro-death autophagyacts as a genuine executioner of, or rather specifically degrades negative regulator(s) of HCD.Of utmost interest in the future is also the need to tackle the question as to how autophagyswitches from pro-life to pro-death mode.

Although it is still uncertain whether autophagy could represent one possible exit of theHCD highway, two other ways of dying have been reported in plants. One relies on VAC-UOLAR PROCESSING ENZYMES (VPE), which encode proteins that share with metacas-pases the typical caspase-hemoglobinase fold (Uren et al., 2000). Tobacco VPE harbour acaspase-1-like activity and are rapidly upregulated in response to TMV. Silencing VPE resultsin abolition of virus-induced hypersensitive lesions associated with the absence of tonoplastdisruption, normally observed and followed by vacuolar collapse in WT plants (Hatsugai et al.,2004). The second way of dying is under the control of the proteasome subunit PBA1 andalso requires vacuole-dependent mechanisms (Hatsugai et al., 2009). Using an RNAi strat-egy targeting PBA1 in Arabidopsis, these authors demonstrated that proteasome-associatedcaspase-3-like activity is necessary for mediating fusion between tonoplast and plasma mem-brane during an incompatible interaction with P. syringae. Interestingly, the vacuolar content



of cells undergoing HCD was also shown to display both anti-bacterial and death-inducing

activities. Overall, these results from the group of Hara-Nishimura illustrate two adaptive

means whereby plant cells can respond to, and dispose of, invaders with respect to pathogen

lifestyle. VPE-controlled vacuolar collapse ending by self-digest of host cells seems to be

employed to cope with intracellular infection by virus whereas PBA1-regulated discharge of

vacuolar content into the apoplasm would be restricted to bacterial pathogens that proliferate

outside of cells. Unfortunately, the proteins targeted by VPE and PBA1 are still unknown.

Besides vacuole-mediated cell deaths, one last highway exit can drive cells to HCD, again in

a plant-specific manner. The corresponding traffic sign indicates a ROS exit. The role of ROS

in signalling–executing plant cell death has long been controversial. This is mainly because

ROS synthesis was considered at first sight to be a by-product of basal metabolism or even as

a consequence of the cell’s demise. In addition, the non-enzymatic origin of some ROS was

difficult to reconcile with the execution of any hypersensitive genetic programmes. However,

combined biochemical and genetic evidence for a role of enzymatic lipid peroxidation in HCD

execution has now been provided. Fatty acid hydroperoxides (FAH) are produced by lipoxyge-

nases (LOX) in response to pathogen attack. Depending on LOX stereo-specificity, molecular

oxygen can be introduced into aliphatic chains on carbon positions 9 or 13 of 18-carbon long

polyunsaturated fatty acids (PUFA), resulting in the formation of 9-FAH or 13-FAH (Blée,

2002). Both types of FAH are potent precursors of numerous oxylipin compounds that are

directly or indirectly involved in defence against pathogens because of their antimicrobial

properties and/or their capability of inducing defence gene expression (Alméras et al., 2003;

Blée, 2002). Nonetheless, the 9-LOX-dependent pathway can also be used for degrading cel-

lular structures.

In many pathosystems, FAH accumulation has been positively correlated with the devel-

opment of hypersensitive symptoms, and vice versa (Cacas et al., 2009; Cacas et al., 2005;

Jalloul et al., 2002; Marmey et al., 2007; Montillet et al., 2005; Rustérucci et al., 1999). Infil-

tration of FAH into tobacco leaves was also shown to mimic HCD in the absence of pathogens

(Rusterucci et al., 1999). Tobacco patatin galactolipases acting upstream of 9-LOX to release

PUFA substrates, mainly from chloroplast membranes, have been identified (Cacas et al., 2005

and 2009; Dhondt et al., 2000). Finally, gain- and loss-of-function approaches carried out

in Arabidopsis targeting patatin genes revealed enhanced and reduced HCD phenotypes in

response to an avirulent strain of P. syringae, respectively (La Camera et al., 2004; La Camera

et al., 2005; La Camera et al., 2009). Anti-sense 9-LOX lines were unable to execute HCD fol-

lowing infection with the oomycete Phytophthora parasitica (Rancé et al., 1998). Even though

the exact mechanisms whereby cells die upon activation of the patatin −9-LOX pathway still

have to be defined, it is likely that: (1) the massive production of cytotoxic FAH would lead to

intense oxidation of proteins, DNA and membranes; and (2) the huge mobilization of plastidial

galactolipids for supplying such an intense oxidative metabolism would result in chloroplast

clearance in cells. Thus, one can assume that these two cytological events would be at the

origin of HCD.

To conclude our trip on the HCD highway, it now seems obvious that plant cells possess

‘more than one way to go’. As previously suggested for animal cells (Wyllie and Golstein,

2001). This means that they can take many distinct itineraries following pathogen recognition.

Depending on the pathogens, entrances to this giant overlapping-highway network are proba-

bly different. Once engaged, they then have to stop by regulatory nodes figured by tolls; some

of these being mandatory, others accessory. They must also avoid any traffic perturbations



occasioned by pathogen effectors. Finally, cells that succeed in going through all these steps

ultimately leave the road web via the appropriate exit.

6.5 ANTIMICROBIAL PROTEINS OR DEFENCE-RELATEDPROTEINS

6.5.1 Introduction

Infection by pathogens is accompanied by the local and systemic induction of plant encoded

proteins referred to as pathogenesis-related (PR) proteins. Initially, PRs were sought for with

passion in different plants treated with various forms of biotic and abiotic stress. Later, they

were formally defined as ‘those proteins that are mostly detectable at basal concentrations in

healthy tissues, but for which accumulation at the protein level has been demonstrated upon

pathological conditions and related situations in at least two or more plant–pathogen com-

binations’ (Van Loon et al., 2006; Van Loon and Van Strien, 1999). The function of PRs in

the resistance of the plant remained uncertain for a long time. However as their various bio-

chemical functions were unveiled, experiments were carried out to test their implication in

the defence of the plant. Besides testing direct antibiotic activity in vitro, many experiments

were carried out using transgenic plants overexpressing given PR genes. Overall, the results

showed that some PRs could provide resistance to certain diseases in certain plants, but in

some combinations no effects were observed. The alternative approach, namely to suppress

PRs, has been hampered by the difficulty that many genes exist for each family of PRs, so

that all forms would need to be suppressed. Some PRs are also expressed constitutively in cer-

tain plant organs, indicating a role in plant development. The conservation during evolution of

PRs both in dicots and in monocots and their occurrence during certain stages of development

or after attack by pathogen, all point towards an important role in plants. Readers interested

in PRs are strongly invited to consult the excellent overview by Van Loon and his colleagues

(2006). More recent specific aspects of PRs have also been recently reviewed (Fernandes et al.,2013; Sels et al., 2008).

6.5.2 Use of PRs for crop protection: Current status

Since PRs have various antifungal and other antibiotic functions their genes have become

popular for designing genetically engineered crops with improved resistance to pathogens.

This has led to a large number of studies (see recent reviews by (Balasubramanian et al., 2012;

Ceasar and Ignacimuthu, 2012; Cletus et al., 2013; Shepherd et al., 2009; Wally et al., 2009).

Many promising results were obtained in laboratory or greenhouse experiments but few data

exist on the performance of such plants under field conditions, currently still a major issue.

In general, the development of transgenic crops has raised some concerns with the pub-

lic, based on reasonable and quite often unreasonable arguments. The fact that this approach

might help to overcome, at least in part, real problems, where so far no chemical approaches

exist, is still much overlooked by the general public. Examples include resistance to nema-

todes, bacteria or viruses that cannot be controlled by conventional pesticides, unlike many

fungal diseases. One such issue is the effectiveness of selected genes in a given crop against

target microorganisms; their expression in the host plants using appropriate promoters can

only be assessed under field conditions that mimic the relevant conditions where such crops



will be used. The development of transgenic crops depends therefore on extensive field trials;in many parts of the world these are not authorized or are only feasible under adverse condi-tions. A recent review by Rommens presents a comprehensive review of the various hurdlesto bringing transgenics to the market (Rommens, 2010). Other issues concerning the use oftransgenic crops expressing PRs is their potential allergenicity (Midoro-Horiuti et al., 2001).This has to be taken into account, for instance, when the transgenic plant is an edible product.Thus a large number of approaches have focused on the use of PRs to improve plant resistanceto pathogens, some of which now await commercial developments.

Finally, other genes might be used to develop resistant crops. They include R genes (resis-tance genes) involved in the detection of effectors and their effects on plant targets. Editingor otherwise inactivating genes for susceptibility might also constitute a possible alternative,once such genes have been identified (Dangl et al., 2013).

6.5.3 Other changes in the transcriptome relatedto pathogenesis

Large scale sequencing technologies that include applications such as genome and transcrip-tome sequencing, mapping of mutations, DNA methylation and histone modifications andalternative splicing or profiling of small RNA profiling, are starting to be increasingly usedin all areas of biology and have brought about a quantum leap in our capacity to acquire newinformation. In the area of induced plant defences, some of these methods are also applied tooffer a real potential for uncovering novel defence pathways, their genes and their proteins,proteins that have hitherto escaped detection. Recent reviews have covered some aspects ofthis (Walley and Dehesh, 2010).

A recent time-course on the transcript accumulation during the interaction between A.thaliana and the bacterial pathogen P. syringae was carried out with RNA-Seq using over500 million read pairs (Howard et al., 2013). The response to infection was analysed in botha susceptible and a resistant host ecotype. Results indicated the induction of a number ofgenes (including PRs such as PR1) consistent with other studies. The most striking resultwas the occurrence of a surprisingly large number of alternative splicing events during thedefence responses of the plant. While a number of genes were known to be alternativelyspliced during defences and were detected in this study, their number greatly exceed whatwas known so far and now provide a new set of candidates the relevance of which can now betested. Alternative splicing in response to stress has also been reviewed recently (Mastrangeloet al., 2012).

The transcriptome of Arabidopsis thaliana, a non-host for Erwinia amylovora, wasanalysed after infection with this bacterium and showed an important re-modelling of thegene transcription. Approximately 20% of induced genes were associated with defence andsignalling. A closer analysis showed that EDS1, a protein of the TIR-NBS-LRR type and2 EDS1-dependent transcription factors, WRKY 46-3 and WRKY 54-2, were identifiedas positive regulators of the defences to E. amylovora. EDS1-independent defences to E.amylovora were also detected in the same study (Moreau et al., 2012).

The early transcriptional changes induced in a resistance faba bean ecotype by Ascochytafabae was studied using genome-wide transcriptome profiling by deepSuperSAGE. Using a2.7-fold change threshold, 1197 sequences were found to be expressed differentially in infectedas compared with control leaves, of which about half were up- and the other down-regulated.A small number of candidates from the SuperSAGE data showing differential expression were



selected and confirmed by qPCR. This comprehensive analysis of the transcriptome of faba

bean in response to A. fabae available provides important new data for further studies in this

important pathosystem (Madrid et al., 2013).

Transcriptional changes were compared between susceptible Vitis vinifera and resistant

Vitis riparia plants after infection by Plasmopara viticola. In general, the difference in resis-

tance between both species is accompanied by a massive increase in transcription rather than

differences in the basal gene expression. At time-points preceding the onset of resistance, new

transcripts coding for PR genes or enzymes of the phenylpropanoid-derived compounds accu-

mulated more strongly in V. riparia than in V. vinifera. The resistance syndrome in V. ripariaalso correlated with the induction of transcripts encoding components of signal transduction

cascades, markers for the hypersensitive reaction and enzymes of jasmonate biosynthesis.

These results might now be exploited to identify candidate genes that are rate-limiting for

deployment of defences and are potentially interesting for genetic engineering of grapevine

resistant to P. viticola (Polesani et al., 2010). The beneficial microorganism Trichodermaharzianum T39 (T39) represents an alternative biological treatment that is used against

P. viticola conventionally controlled using fungicides. The transcriptome of control and

T39-treated V. vinifera ‘Pinot noir’ plants were compared after infection by P. viticola using

next-generation RNA sequencing (RNAseq). The induction of resistance was accompanied by

the differential expression of 7024 genes and treatments with T39-induced genes connected

with the recognition of microbes or processes related to defence that correlated with the

resistance induced by T39 in the susceptible grapevine variety. These results now offer the

possibility to isolate candidates useful for enhancing resistance of grapevine, to identify sets

of markers to be used in the selection and development of new inducers of resistance or to

optimize the use of inducers of resistance (Perazzolli et al., 2012).

Follow-ups of results from genome-wide gene expression analyses require further studies

to determine the relative importance of some of the identified genes. This may be much

more complicated, since a process such as induced resistance is very likely to depend on

combinations of genes. This was recently highlighted by a study in tomato treated with

acibenzolar-S-methyl (ASM, commercialized as Actigard® by Syngenta Inc.), a chemical

that induces defence responses similar to those activated during systemic acquired resistance

(Friedrich et al., 1996; Zuluaga et al., 2013). While the microarray survey nicely identified

genes that were reliably detected in all replications and correlated well with resistance

suppression, silencing of only a small number of them did not show any reduction in the

ASM-induced resistance (Zuluaga et al., 2013).

RNAseq was used on susceptible lettuce after inoculation with the compatible necrotrophic

fungal pathogen Botrytis cinerea. Genes belonging to the phenylpropanoid, the terpenoid path-

ways, and abscisic acid-dependent genes were induced, whereas photosynthesis-related genes

were downregulated. The gene expression in lettuce responding to B. cinerea correlated with

that of lettuce infected by the compatible downy mildew pathogen Bremia lactucae. Inter-

estingly, there is an overlap in the transcriptome of the compatible interaction of lettuce to

necrotrophic or biotrophic pathogens. The expression of lettuce genes involved in signalling

pathways in these compatible pathogen interactions corresponded to that observed in a com-

patible interaction between maize and Ustilago maydis (Doehlemann et al., 2008). All these

findings might eventually lead to an identification of genes that are targeted or modified by

compatible pathogens leading to a susceptible state or to resistance mechanisms that are pre-

vented by the true pathogens (De Cremer et al., 2013).



6.6 CONCLUSIONS

With the new advances in various large-scale analyses, our knowledge of plant defences is

rapidly expanding. However, many things are still not understood. Resistance is clearly the

product of a complex combination of reactions and their hierarchical relations require time

to disentangle. The reactions taking place in compatible plant–pathogen interactions are also

increasingly being studied as they might lead us to new disease resistance mechanisms but also

to potential targets in the pathogen. A comprehensive representation of the defence responses

of a plant to a pathogen should also take into account environmental conditions. The sig-

nificance of acquiring more knowledge, for example on the behaviour of transgenic lines in

the field, is of prime importance for some practical applications that could progress through

our knowledge. Finally, more than ever this general area of research offers many interesting

opportunities for basic and applied research.

REFERENCES

Ahuja I, Kissen R, Bones AM, 2012. Phytoalexins in defense against pathogens. Trends in Plant Science 17,73–90.

Aist JR, 1976. Papillae and related wound plugs of plant cells. Annual Review of Phytopathology 14, 145–163.

Alamillo JM, García-Olmedo F, 2001. Effects of urate, a natural inhibitor of peroxynitrite-mediated toxicity,in the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae. The Plant Journal25, 529–540.

Alméras E, Stolz S, Vollenweider S, Reymond P, Mène-Saffrané L, Farmer EE, 2003. Reactive electrophilespecies activate defense gene expression in Arabidopsis. The Plant Journal 34, 205–216.

Assaad FF, Qiu JL, Youngs H, Ehrhardt D, Zimmerli L, Kalde M, Wanner G, Peck SC, Edwards H, Ramonell K,Somerville CR, Thordal-Christensen H, 2004. The PEN1 syntaxin defines a novel cellular compartmentupon fungal attack and is required for the timely assembly of papillae. Molecular Biology of the Cell 15,5118–5129.

Asselbergh B, Höfte M, 2007. Basal tomato defences to Botrytis cinerea include abscisic acid-dependent cal-lose formation. Physiological and Molecular Plant Pathology 71, 33–40.

Balasubramanian V, Vashisht D, Cletus J, Sakthivel N, 2012. Plant β-1,3-glucanases: their biological functionsand transgenic expression against phytopathogenic fungi. Biotechnology Letters 34, 1983–1990.

Barabote RD, Johnson OL, Zetina E, San Francisco SK, Fralick JA, San Francisco MJD, 2003. Erwiniachrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopatho-genesis. Journal of Bacteriology 185, 5772–5778.

Bednarek P, 2012. Chemical warfare or modulators of defence responses - the function of secondary metabo-lites in plant immunity. Current Opinion in Plant Biology 15, 407–414.

Bednarek P, Schneider B, Svatos A, Oldham NJ, Hahlbrock K, 2005. Structural complexity, differentialresponse to infection, and tissue specificity of indolic and phenylpropanoid secondary metabolism in Ara-bidopsis roots. Plant Physiology 138, 1058–1070.

Bednarek P, Osbourn A, 2009. Plant-microbe interactions: chemical diversity in plant defense. Science 324,746–748.

Bellin D, Asai S, Delledonne M, Yoshioka H, 2013. Nitric oxide as a mediator for defense responses. MolecularPlant-Microbe Interactions 26, 271–277.

Belofsky G, Percivill D, Lewis K, Tegos GP, Ekart J, 2004. Phenolic metabolites of Dalea versicolor thatenhance antibiotic activity against model pathogenic bacteria. Journal of Natural Products 67, 481–484.

Bent A, 2011. Pathogens drop the hint: don’t forget phytoalexin pathways. Cell Host and Microbe 9, 169–170.

Berry DL, Baehrecke EH, 2007. Growth arrest and autophagy are required for salivary gland cell degradationin Drosophila. Cell 131, 1137–1148.

Besson-Bard A, Courtois C, Gauthier A, Dahan J, Dobrowolska G, Jeandroz S, Pugin A, Wendehenne D, 2008.Nitric oxide in plants: production and cross-talk with Ca2+ signaling. Molecular Plant 1, 218–228.



Bestwick CS, Brown IR, Bennett MH, Mansfield JW, 1997. Localization of hydrogen peroxide accumulationduring the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. The Plant Cell9, 209–221.

Bhuiyan NH, Selvaraj G, Wei YD, King J, 2009. Gene expression profiling and silencing reveal that monolignolbiosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion. Journalof Experimental Botany 60, 509–521.

Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C,Davies DR, Ausubel FM, Bolwell GP, 2006. Peroxidase-dependent apoplastic oxidative burst in Arabidopsisrequired for pathogen resistance. The Plant Journal 47, 851–863.

Birkenbihl RP, Diezel C, Somssich IE, 2012. Arabidopsis WRKY33 is a key transcriptional regulator of hor-monal and metabolic responses toward Botrytis cinerea infection. Plant Physiology 159, 266–285.

Blanvillain R, Young B, Cai Y-m, Hecht V, Varoquaux F, Delorme V, Lancelin J-M, Delseny M, Gallois P,2011. The Arabidopsis peptide kiss of death is an inducer of programmed cell death. EMBO Journal 30,1173–1183.

Blée E, 2002. Impact of phyto-oxylipins in plant defense. Trends in Plant Science 7, 315–322.Blee K, Jupe S, Richard G, Zimmerlin A, Davies D, Bolwell GP, 2001. Molecular identification and expression

of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and relatedmembers of the gene family. Plant Molecular Biology 47, 607–620.

Boerjan W, Ralph J, Baucher M, 2003. Lignin biosynthesis. Annual Review of Plant Biology 54, 519–546.Bohlenius H, Morch SM, Godfrey D, Nielsen ME, Thordal-Christensen H, 2010. The multivesicular

body-localized GTPase ARFA1b/1c is important for callose deposition and ror2 syntaxin-dependent prein-vasive basal defense in barley. Plant Cell 22, 3831–3844.

Bosis E, Salomon D, Sessa G, 2011. A simple yeast-based strategy to identify host cellular processes targetedby bacterial effector proteins. PloS ONE 6, e27698.

Bourque S, Dutartre A, Hammoudi V, Blanc S, Dahan J, Jeandroz S, Pichereaux C, Rossignol M, Wende-henne D, 2011. Type-2 histone deacetylases as new regulators of elicitor-induced cell death in plants. NewPhytologist 192, 127–139.

Burse A, Weingart H, Ullrich MS, 2004. The phytoalexin-inducible multidrug efflux pump AcrAB contributesto virulence in the fire blight pathogen, Erwinia amylovora. Molecular Plant-Microbe Interactions 17,43–54.

Burton RA, Gidley MJ, Fincher GB, 2010. Heterogeneity in the chemistry, structure and function of plant cellwalls. Nature Chemical Biology 6, 724–732.

Cacas J-L, 2010. Devil inside: does plant programmed cell death involve the endomembrane system? Plant,Cell and Environment 33, 1453–1473.

Cacas J-L, Vailleau F, Davoine C, Ennar N, Agnel J-P, Tronchet M, Ponchet M, Blein J-P, Roby D, Trianta-phylides C, Montillet J-L, 2005. The combined action of 9 lipoxygenase and galactolipase is sufficient tobring about programmed cell death during tobacco hypersensitive response. Plant, Cell and Environment28, 1367–1378.

Cacas J-L, Marmey P, Montillet J-L, Sayegh-Alhamdia M, Jalloul A, Rojas-Mendoza A, Clérivet A, NicoleM, 2009. A novel patatin-like protein from cotton plant, GhPat1, is co-expressed with GhLox1 during Xan-thomonas campestris-mediated hypersensitive cell death. Plant Cell Reports 28, 155–164.

Carver TLW, Zeyen RJ, Robbins MP, Dearne GA, 1992. Effects of the PAL inhibitor, AOPP, on oat, bar-ley and wheat cell responses to appropriate and inappropriate formae specialis of Erysiphe graminis DC.Physiological and Molecular Plant Pathology 41, 397–409.

Ceasar SA, Ignacimuthu S, 2012. Genetic engineering of crop plants for fungal resistance: role of antifungalgenes. Biotechnology Letters 34, 995–1002.

Chae H-J, Ke N, Kim H-R, Chen S, Godzik A, Dickman M, Reed JC, 2003. Evolutionarily conserved cytopro-tection provided by Bax inhibitor-1 homologs from animals, plants, and yeast. Gene 323, 101–113.

Chaouch S, Queval G, Noctor G, 2012. AtRbohF is a crucial modulator of defence-associated metabolism anda key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis.The Plant Journal 69, 613–627.

Chizzali C, Beerhues L, 2012. Phytoalexins of the Pyrinae: Biphenyls and dibenzofurans. Beilstein Journal ofOrganic Chemistry 8, 613–620.

Ciuffetti LM, VanEtten HD, 1996. Virulence of a pisatin demethylase-deficient Nectria haematococca MPVIisolate is increased by transformation with a pisatin demethylase gene. Molecular Plant-Microbe Interac-tions 9, 787–792.



Clarke A, Desikan R, Hurst RD, Hancock JT, Neill SJ, 2000. NO way back: nitric oxide and programmed celldeath in Arabidopsis thaliana suspension cultures. 24, 667–677.

Clay NK, Adio AM, Denoux C, Jander G, Ausubel FM, 2009. Glucosinolate metabolites required for an ara-bidopsis innate immune response. Science 323, 95–101.

Cletus J, Balasubramanian V, Vashisht D, Sakthivel N, 2013. Transgenic expression of plant chitinases toenhance disease resistance. Biotechnology Letters 35, 1719–1732.

Clough SJ, Fengler KA, Yu Ic, Lippok B, Smith RK, Bent AF, 2000. The Arabidopsis DND1 “defense, nodeath” gene encodes a mutated cyclic nucleotide-gated ion channel. Proceedings of the National Academyof Sciences USA 97, 9323–9328.

Coleman JJ, White GJ, Rodriguez-Carres M, VanEtten HD, 2011. An ABC transporter and a cytochrome P450of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to thephytoalexin pisatin. Molecular Plant-Microbe Interactions 24, 368–376.

Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl JL, Epple P, 2010. Arabidopsis type 1metacaspases control cell death. Science 330, 1393–1397.

Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Hückelhoven R, Stein M, Freialden-hoven A, Somerville SC, Schulze-Lefert P, 2003. SNARE-protein-mediated disease resistance at the plantcell wall. Nature 425, 973–977.

Cooper RM, Resende MLV, Flood J, Rowan MG, Beale MH, Potter U, 1996. Detection and cellular localizationof elemental sulphur in disease-resistant genotypes of Theobroma cacao. Nature 379, 159–162.

Cui F, Brosché M, Sipari N, Tang S, Overmyer K, 2013. Regulation of ABA dependent wound induced spread-ing cell death by MYB108. New Phytologist 200, 634–640.

Daayf F, Schmitt A, Bélanger RR. 1997. Evidence of phytoalexins in cucumber leaves infected with powderymildew following treatment with leaf extracts of Reynoutria sachalinensis. Plant Physiology 113, 719–727.

Dangl JL, Jones JDG, 2001. Plant pathogens and integrated defence responses to infection. Nature 411,826–833.

Dangl JL, Horvath DM, Staskawicz BJ, 2013. Pivoting the plant immune system from dissection to deployment.Science 341, 746–751.

Danon A, Rotari VI, Gordon A, Mailhac N, Gallois P, 2004. Ultraviolet-C overexposure induces programmedcell death in Arabidopsis, which is mediated by caspase-like activities and which can be suppressed by Cas-pase inhibitors, p35 and Defender Against Apoptotic Death. Journal of Biological Chemistry 279, 779–787.

Dat JF, Pellinen R, Tom B, Van De Cotte B, Langebartels C, Kangasjärvi J, Inzé D, Van Breusegem F, 2003.Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. The Plant Journal33, 621–632.

Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP, 2012. The apoplasticoxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. The PlantCell Online 24, 275–287.

Day B, Henty JL, Porter KJ, Staiger CJ, 2011. The pathogen-actin connection: a platform for defense signalingin plants. Annual Review of Phytopathology, 49, 483–506.

De Cremer K, Mathys J, Vos C, Froenicke L, Michelmore RW, Cammue BPA, De Coninck B, 2013.RNAseq-based transcriptome analysis of Lactuca sativa infected by the fungal necrotroph Botrytis cinerea.Plant Cell and Environment 36, 1992–2007.

de Pinto MC, Tommasi F, De Gara L, 2002. Changes in the antioxidant systems as part of the signaling pathwayresponsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobaccoBright-Yellow 2 Cells. Plant Physiology 130, 698–708.

de Pinto MC, Paradiso A, Leonetti P, De Gara L, 2006. Hydrogen peroxide, nitric oxide and cytosolic ascorbateperoxidase at the crossroad between defence and cell death. The Plant Journal 48, 784–795.

de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW, Egea PR, Bogre L, Grant M, 2007.Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease.Embo Journal 26, 1434–1443.

Delledonne M, Xia Y, Dixon RA, Lamb C, 1998. Nitric oxide functions as a signal in plant disease resistance.Nature 394, 585–588.

Delledonne M, Zeier J, Marocco A, Lamb C, 2001. Signal interactions between nitric oxide and reactive oxygenintermediates in the plant hypersensitive disease resistance response. Proceedings of the National Academyof Sciences USA 98, 13454–13459.

Desikan R, A.-H.-Mackerness S, Hancock JT, Neill SJ, 2001. Regulation of the Arabidopsis transcriptome byoxidative stress. Plant Physiology 127, 159–172.



Dhondt S, Geoffroy P, Stelmach BA, Legrand M, Heitz T, 2000. Soluble phospholipase A2 activity isinduced before oxylipin accumulation in tobacco mosaic virus-infected tobacco leaves and is contributedby patatin-like enzymes. The Plant Journal 23, 431–440.

Dixon RA, Paiva NL, 1995. Stress-induced phenylpropanoid metabolism. The Plant Cell 7, 1085–1097.Dixon RA, Harrison MJ, Paiva NL, 1995. The isoflavonoid phytoalexin pathway: From enzymes to genes to

transcription factors. Physiologia Plantarum 93, 385–392.Doehlemann G, Wahl R, Horst RJ, Voll LM, Usadel B, Poree F, Stitt M, Pons-Kuhnemann J, Sonnewald U,

Kahmann R, Kamper J, 2008. Reprogramming a maize plant: transcriptional and metabolic changes inducedby the fungal biotroph Ustilago maydis. The Plant Journal 56, 181–195.

Doke N, 1983. Generation of superoxide anion by potato tuber protoplasts during the hypersensitive responseto hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressorsof hypersensitivity. Physiological Plant Pathology 23, 359–367.

Du Fall LA, Solomon PS, 2013. The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexinin wheat. New Phytologist 200, 185–200.

Ellinger D, Naumann M, Falter C, Zwikowics C, Jamrow T, Manisseri C, Somerville SC, Voigt CA, 2013. Ele-vated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis.Plant Physiology 161, 1433–1444.

Ettefagh KA, Burns JT, Junio HA, Kaatz GW, Cech NB, 2011. Goldenseal (Hydrastis canadensis L.) extractssynergistically enhance the antibacterial activity of berberine via efflux pump inhibition. Planta Medica 77,835–840.

Fan X, Mattheis JP, Roberts RG, 2000. Biosynthesis of phytoalexin in carrot root requires ethylene action.Physiologia Plantarum 110, 450–454.

Fernandes H, Michalska K, Sikorski M, Jaskolski M, 2013. Structural and functional aspects of PR-10 proteins.FEBS Journal 280, 1169–1199.

Fiamegos YC, Kastritis PL, Exarchou V, Han H, Bonvin AMJJ, Vervoort J, Lewis K, Hamblin MR, Tegos GP,2011. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthiumagainst gram-positive pathogenic bacteria. PLoS ONE 6, e18127.

Field B, Fiston-Lavier A-S, Kemen A, Geisler K, Quesneville H, Osbourn AE, 2011. Formation of plantmetabolic gene clusters within dynamic chromosomal regions. Proceedings of the National Academy ofSciences USA 108, 16116–16121.

Fleissner A, Sopalla C, Weltring KM, 2002. An ATP-binding cassette multidrug-resistance transporter is nec-essary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers. MolecularPlant-Microbe Interactions 15, 102–108.

Flores-Sanchez IJ, Verpoorte R, 2009. Plant polyketide synthases: a fascinating group of enzymes. Plant Phys-iology and Biochemistry 47, 167–174.

Fofana B, Benhamou N, McNally DJ, Labbé C, Séguin A, Bélanger RR, 2005. Suppression of induced resis-tance in cucumber through disruption of the flavonoid pathway. Phytopathology 95, 114–123.

Friedrich L, Lawton K, Ruess W, Masner P, Specker N, Rella MG, Meier B, Dincher S, Staub T, Uknes S,Metraux JP, Kessmann H, Ryals J, 1996. A benzothiadiazole derivative induces systemic acquired resistancein tobacco. The Plant Journal 10, 61–70.

Gaid MM, Sircar D, Müller A, Beuerle T, Liu B, Ernst L, Hänsch R, Beerhues L, 2012. Cinnamate:CoA LigaseInitiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.Plant Physiology 160, 1267–1280.

Gaupels F, Spiazzi-Vandelle E, Yang D, Delledonne M, 2011. Detection of peroxynitrite accumulation inArabidopsis thaliana during the hypersensitive defense response. Nitric Oxide Biology and Chemistry 25,222–228.

Gechev T, Minkov I, Hille J, 2005. Hydrogen peroxide-induced cell death in Arabidopsis: transcriptional andmutant analysis reveals a role of an oxoglutarate-dependent dioxygenase gene in the cell death process.IUBMB Life 57, 181–188.

Giorcelli A, Sparvoli F, Mattivi F, Tava A, Balestrazzi A, Vrhovsek U, Calligari P, Bollini R, Confalonieri M,2004. Expression of the stilbene synthase (StSy) gene from grapevine in transgenic white poplar results inhigh accumulation of the antioxidant resveratrol glucosides. Transgenic Research 13, 203–214.

Gleason C, Huang S, Thatcher LF, Foley RC, Anderson CR, Carroll AJ, Millar AH, Singh KB, 2011.Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence onplant stress gene regulation and defense. Proceedings of the National Academy of Sciences USA 108,10768–10773.



González-Lamothe R, Mitchell G, Gattuso M, Diarra MS, Malouin F, Bouarab K, 2009. Plant antimicrobialagents and their effects on plant and human pathogens. International Journal of Molecular Sciences 10,3400–3419.

Goodman N, Novacky AJ, 1994. The Hypersensitive Reaction in Plants to Pathogens. A Resistance Phe-nomenon. St Paul, MN: APS Press edn.

Graham TL, Graham MY, Subramanian S, Yu O, 2007. RNAi silencing of genes for elicitation or biosynthesisof 5-deoxyisoflavonoids suppresses race-specific resistance and hypersensitive cell death in Phytophthorasojae infected tissues. Plant Physiology 144, 728–740.

Grant JJ, Loake GJ, 2000. Role of reactive oxygen intermediates and cognate redox signaling in disease resis-tance. Plant Physiology 124, 21–30.

Grayer RJ, Kokubun T, 2001. Plant-fungal interactions: the search for phytoalexins and other antifungal com-pounds from higher plants. Phytochemistry 56, 253–263.

Hahn MG, Bonhoff A, Grisebach H, 1985. Quantitative localization of the phytoalexin glyceollin in relation tofungal hyphae in soybean roots infected with Phytophthora megasperma f. sp. glycinea. Plant Physiology77, 591–601.

Hain R, Reif HJ, Krause E, Langebartels R, Kindl H, Vornam B, Wiese W, Schmelzer E, Schreier PH, StöckerRH, 1993. Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 361,153–156.

Harborne JB, 1999. The comparative biochemistry of phytoalexin induction in plants. Biochemical Systematicsand Ecology 27, 335–367.

Hardham AR, Jones DA, Takemoto D, 2007. Cytoskeleton and cell wall function in penetration resistance.Current Opinion in Plant Biology 10, 342–348.

Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M, Nishimura M, Hara-Nishimura I, 2004.A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science 305, 855–858.

Hatsugai N, Iwasaki S, Tamura K, Kondo M, Fuji K, Ogasawara K, Nishimura M, Hara-Nishimura I, 2009. Anovel membrane fusion-mediated plant immunity against bacterial pathogens. Genes and Development 23,2496–2506.

He XZ, Dixon RA, 2000. Genetic manipulation of isoflavone 7-O-methyltransferase enhances biosynthesis of4′-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. Plant Cell 12, 1689–1702.

Heitefuss R, 2001. Defence reactions of plants to fungal pathogens: principles and perspectives, using powderymildew on cereals as an example. Naturwissenschaften 88, 273–283.

Hemmerlin A, Harwood JL, Bach TJ, 2012. A raison d’être for two distinct pathways in the early steps of plantisoprenoid biosynthesis? Progress in Lipid Research 51, 95–148.

Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI, Petersen NHT, Mattsson O, Jørgensen LB, Jones JDG,Mundy J, Petersen M, 2009. Autophagic Components contribute to hypersensitive cell death in Arabidopsis.Cell 137, 773–783.

Hoisington DA, Neuffer MG, Walbot V, 1982. Disease lesion mimics in maize: I. Effect of genetic background,temperature, developmental age, and wounding on necrotic spot formation with Les1. Developmental Biol-ogy 93, 381–388.

Howard BE, Hu Q, Babaoglu AC, Chandra M, Borghi M, Tan X, He L, Winter-Sederoff H, GassmannW, Veronese P, Heber S, 2013. High-throughput RNA sequencing of Pseudomonas-infected Arabidopsisreveals hidden transcriptome complexity and novel splice variants. PLoS ONE 8, e74183.

Hückelhoven R, 2004. BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryoticrelatives. Apoptosis 9, 299–307.

Hückelhoven R, 2007. Cell wall - Associated mechanisms of disease resistance and susceptibility. AnnualReview of Phytopathology 45, 101–127.

Hückelhoven R, Fodor J, Preis C, Kogel K-H. 1999. Hypersensitive cell death and papilla formation in barleyattacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acidaccumulation. Plant Physiology 119, 1251–1260.

Huffaker A, Kaplan F, Vaughan MM, Dafoe NJ, Ni X, Rocca JR, Alborn HT, Teal PEA, Schmelz EA, 2011.Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. PlantPhysiology 156, 2082–2097.

Humphreys JM, Chapple C, 2002. Rewriting the lignin roadmap. Current Opinion in Plant Biology 5,224–229.

Ibraheem F, Gaffoor I, Chopra S. 2010. Flavonoid phytoalexin-dependent resistance to anthracnose leaf blightrequires a functional yellow seed1 in Sorghum bicolor. Genetics 184, 915–926.



Inoue Y, Sakai M, Yao Q, Tanimoto Y, Toshima H, Hasegawa M, 2013. Identification of a novel casbane-typediterpene phytoalexin, ent-10-oxodepressin, from rice leaves. Bioscience Biotechnology and Biochemistry77, 760–765.

Jacobs AK, Lipka V, Burton RA, Panstruga R, Strizhov N, Schulze-Lefert P, Fincher GB, 2003. An Arabidopsiscallose synthase, GSL5, is required for wound and papillary callose formation. Plant Cell 15, 2503–2513.

Jalloul A, Montillet JL, Assigbetsé K, Agnel JP, Delannoy E, Triantaphylidès C, Daniel JF, Marmey P, GeigerJP, Nicole M, 2002. Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenasesduring the hypersensitive reaction. The Plant Journal 32, 1–12.

Jeandet P, Delaunois B, Conreux A, Donnez D, Nuzzo V, Cordelier S, Clément C, Courot E, 2010. Biosyn-thesis, metabolism, molecular engineering, and biological functions of stilbene phytoalexins in plants.BioFactors 36, 331–341.

Jeandet P, Clément C, Courot E, Cordelier S, 2013. Modulation of phytoalexin biosynthesis in engineeredplants for disease resistance. International Journal of Molecular Science 14, 14136–14170.

Jones JDG, Dangl JL, 2006. The plant immune system. Nature 444, 323–329.Jørgensen K, Rasmussen AV, Morant M, Nielsen AH, Bjarnholt N, Zagrobelny M, Bak S, Møller BL, 2005.

Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Current Opin-ion in Plant Biology 8, 280–291.

Kalia NP, Mahajan P, Mehra R, Nargotra A, Sharma JP, Koul S, Khan IA, 2012. Capsaicin, a novel inhibitor ofthe NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus. Journal of AntimicrobialChemotherapy 67, 2401–2408.

Khan R, Tan R, Mariscal AG, Straney D, 2003. A binuclear zinc transcription factor binds the hostisoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification. MolecularMicrobiology 49, 117–130.

Klempien A, Kaminaga Y, Qualley A, Nagegowda DA, Widhalm JR, Orlova I, Shasany AK, Taguchi G, KishCM, Cooper BR, D’Auria JC, Rhodes D, Pichersky E, Dudareva N, 2012. Contribution of CoA ligases tobenzenoid biosynthesis in petunia flowers. Plant Cell 24, 2015–2030.

Kliebenstein DJ, 2004. Secondary metabolites and plant/environment interactions: a view through Arabidopsisthaliana tinged glasses. Plant, Cell and Environment 27, 675–684.

Kliebenstein DJ, Rowe HC, Denby KJ, 2005. Secondary metabolites influence Arabidopsis/Botrytis interac-tions: variation in host production and pathogen sensitivity. The Plant Journal 44, 25–36.

Kobayashi S, Ding CK, Nakamura Y, Nakajima I, Matsumoto R, 2000. Kiwifruits (Actinidia deliciosa) trans-formed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside). Plant Cell Reports 19,904–910.

Kogel KH, Beckhove U, Dreschers J, Munch S, Romme Y, 1994. Acquired-resistance in barley - the resis-tance mechanism induced by 2,6-dichloroisonicotinic acid is a phenocopy of a genetically based mechanismgoverning race-specific powdery mildew resistance. Plant Physiology 106, 1269–1277.

Kuc J, 1995. Phytoalexins, stress metabolism, and disease resistance in plants. Annual Review of Phytopathol-ogy 33, 275–297.

Kurata N, Miyoshi K, Nonomura K-I, Yamazaki Y, Ito Y, 2005. Rice mutants and genes related to organdevelopment, morphogenesis and physiological traits. Plant and Cell Physiology 46, 48–62.

Kurosaki F, 1994. Regulation of biosynthesis of carrot phytoalexin 6-methoxymellein. Phytochemistry 37,727–730.

La Camera S, Gouzerh G, Dhondt S, Hoffmann L, Fritig B, Legrand M, Heitz T, 2004. Metabolic reprogram-ming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways. ImmunologicalReviews 198, 267–284.

La Camera S, Geoffroy P, Samaha H, Ndiaye A, Rahim G, Legrand M, Heitz T, 2005. A pathogen-induciblepatatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis. The PlantJournal 44, 810–825.

La Camera S, Balague C, Gobel C, Geoffroy P, Legrand M, Feussner I, Roby D, Heitz T, 2009. The Arabidop-sis patatin-like protein 2 (PLP2) plays an essential role in cell death execution and differentially affectsbiosynthesis of oxylipins and resistance to pathogens. Molecular Plant-Microbe Interactions 22, 469–481.

Laloi C, Apel K, Danon A, 2004. Reactive oxygen signalling: the latest news. Current Opinion in Plant Biology7, 323–328.

Lamotte O, Gould K, Lecourieux D, Sequeira-Legrand A, Lebrun-Garcia A, Durner J, Pugin A, WendehenneD, 2004. Analysis of nitric oxide signaling functions in tobacco cells challenged by the elicitor cryptogein.Plant Physiology 135, 516–529.



Larbat R, Hehn A, Hans J, Schneider S, Jugdé H, Schneider B, Matern U, Bourgaud F, 2009. Isolation andfunctional characterization of CYP71AJ4 Encoding for the first P450 monooxygenase of angular fura-nocoumarin biosynthesis. Journal of Biological Chemistry 284, 4776–4785.

Lewis K, Ausubel FM, 2006. Prospects for plant-derived antibacterials. Nature Biotechnology 24, 1504–1507.Lewis NG, Davin LB, 1999. Lignans: Biosynthesis and function. In: Derek B, Koji N, eds. Comprehensive

Natural Products Chemistry. Oxford: Pergamon, pp. 639–712.Liang P-H, Ko T-P, Wang AHJ, 2002. Structure, mechanism and function of prenyltransferases. European

Journal of Biochemistry 269, 3339–3354.Lipka V, Panstruga R, 2005. Dynamic cellular responses in plant-microbe interactions. Current Opinion in

Plant Biology 8, 625–631.Lipka V, Kwon C, Panstruga R, 2007. SNARE-Ware: The role of SNARE-domain proteins in plant biology.

Annual Review of Cell and Developmental Biology 23, 147–174.Liu H, Du Y, Chu H, Shih CH, Wong YW, Wang M, Chu IK, Tao Y, Lo C, 2010. Molecular dissection of the

pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum. Plant and Cell Physiology 51,1173–1185.

Liu Y, Schiff M, Czymmek K, Tallóczy Z, Levine B, Dinesh-Kumar SP, 2005. Autophagy regulates pro-grammed cell death during the plant innate immune response. Cell 121, 567–577.

Lorrain S, Vailleau F, Balagué C, Roby D, 2003. Lesion mimic mutants: keys for deciphering cell death anddefense pathways in plants? Trends in Plant Science 8, 263–271.

Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J, 2010. Callose deposition: a multifaceted plantdefense response. Molecular Plant-Microbe Interactions 24, 183–193.

Madrid E, Palomino C, Plotner A, Horres R, Rotter B, Winter P, Krezdorn N, Torres AM, 2013. DeepSuper-Sage analysis of the Vicia faba transcriptome in response to Ascochyta fabae infection. PhytopathologiaMediterranea 52, 166–182.

Marmey P, Jalloul A, Alhamdia M, Assigbetse K, Cacas J-L, Voloudakis AE, Champion A, Clerivet A, Montil-let J-L, Nicole M, 2007. The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction ofcotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum. Plant Physiology and Biochem-istry 45, 596–606.

Mastrangelo AM, Marone D, Laido G, De Leonardis AM, De Vita P, 2012. Alternative splicing: Enhancingability to cope with stress via transcriptome plasticity. Plant Science 185, 40–49.

Matern U, Lüer P, Kreusch D, 1999. Biosynthesis of Coumarins. In: Derek B, Koji N, eds. ComprehensiveNatural Products Chemistry. Oxford: Pergamon, pp. 623–637.

McLellan H, Boevink PC, Armstrong MR, Pritchard L, Gomez S, Morales J, Whisson SC, Beynon JL, BirchPRJ, 2013. An RxLR Effector from Phytophthora infestans prevents re-localisation of two plant nac tran-scription factors from the endoplasmic reticulum to the nucleus. PLoS Pathogens 9, e1003670.

McLusky SR, Bennett MH, Beale MH, Lewis MJ, Gaskin P, Mansfield JW, 1999. Cell wall alterations andlocalized accumulation of feruloyl-3′-methoxytyramine in onion epidermis at sites of attempted penetrationby Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoidbiosynthesis. The Plant Journal 17, 523–534.

Midoro-Horiuti T, Brooks EG, Goldblum RM, 2001. Pathogenesis-related proteins of plants as allergens.Annals of Allergy, Asthma and Immunology 87, 261–271.

Milani NA, Lawrence DP, Arnold AE, VanEtten HD, 2012. Origin of pisatin demethylase (PDA) in the genusFusarium. Fungal Genetics and Biology 49, 933–942.

Millar AH, Day DA, 1996. Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plantmitochondria. FEBS Letters 398, 155–158.

Mittler R, Herr EH, Orvar BL, van Camp W, Willekens H, Inzé D, Ellis BE, 1999. Transgenic tobacco plantswith reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection.Proceedings of the National Academy of Sciences USA 96, 14165–14170.

Moersbacher B, Mendgen K, 2000. Structural aspects of defense. In: Van Loon LC, Fraser RSS, eds. Mecha-nisms of Resistance to Plant Diseases. Dordrecht: Kluwer, pp. 231–277.

Montillet J-L, Chamnongpol S, Rustérucci C, Dat J, van de Cotte B, Agnel J-P, Battesti C, Inzé D, VanBreusegem F, Triantaphylidès C, 2005. Fatty acid hydroperoxides and H2O2 in the execution of hyper-sensitive cell death in tobacco leaves. Plant Physiology 138, 1516–1526.

Moreau M, Degrave A, Vedel R, Bitton F, Patrit O, Renou J-P, Barny M-A, Fagard M, 2012. EDS1 Con-tributes to nonhost resistance of Arabidopsis thaliana against Erwinia amylovora. Molecular Plant-MicrobeInteractions 25, 421–430.



Morel C, Stermitz FR, Tegos G, Lewis K, 2003. Isoflavones as potentiators of antibacterial activity. Journal ofAgricultural and Food Chemistry 51, 5677–5679.

Morel JB, Dangl JL, 1997. The hypersensitive response and the induction of cell death in plants. Cell Deathand Differentiation 4, 671–683.

Mühlenbock P, Szechynska-Hebda M, Płaszczyca M, Baudo M, Mateo A, Mullineaux PM, Parker JE,Karpinska B, Karpinski S, 2008. Chloroplast signaling and LESION SIMULATING DISEASE1 regulatecrosstalk between light acclimation and immunity in Arabidopsis. Plant Cell 20, 2339–2356.

Müller KO, Börger H, 1940. Experimentelle Untersuchungen über die Phytophtora-Resistenz der Kartoffel.Arbeiten aus der Biologischen Reichanstalt für Land- und Forstwirtschaft 23, 189–231.

Nawar HF, Kuti JO, 2003. Wyerone acid phytoalexin synthesis and peroxidase activity as markers for resistanceof broad beans to chocolate spot disease. Journal of Phytopathology 151, 564–570.

Neill SJ, Desikan R, Hancock JT, 2003. Nitric oxide signalling in plants. New Phytologist 159, 11–35.Neilson EH, Goodger JQD, Woodrow IE, Moller BL, 2013. Plant chemical defense: at what cost? Trends in

Plant Science 18, 250–258.Nicholson RL, Hammerschmidt R, 1992. Phenolic compounds and their role in disease resistance. Annual

Review of Phytopathology 30, 369–389.Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC, 2003. Loss of a callose synthase

results in salicylic acid-dependent disease resistance. Science 301, 969–972.O’Brien JA, Daudi A, Butt VS, Bolwell GP, 2012a. Reactive oxygen species and their role in plant defence

and cell wall metabolism. Planta 236, 765–779.O’Brien JA, Daudi A, Finch P, Butt VS, Whitelegge JP, Souda P, Ausubel FM, Bolwell GP, 2012b. A

peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-eliciteddefense. Plant Physiology 158, 2013–2027.

Okada K, 2011. The biosynthesis of isoprenoids and the mechanisms regulating it in plants. Bioscience Biotech-nology and Biochemistry 75, 1219–1225.

Ostergaard L, Petersen M, Mattsson O, Mundy J, 2002. An Arabidopsis callose synthase. Plant MolecularBiology 49, 559–566.

Ostertag M, Stammler J, Douchkov D, Eichmann R, Hückelhoven R, 2013. The conserved oligomeric Golgicomplex is involved in penetration resistance of barley to the barley powdery mildew fungus. MolecularPlant Pathology 14, 230–240.

Patel S, Dinesh-Kumar SP, 2008. Arabidopsis ATG6 is required to limit the pathogen-associated cell deathresponse. Autophagy 4, 20–27.

Paxton JD, 1981. Phytoalexins – a working redefinition. Journal of Phytopathology 101, 106–109.Pedras MSC, Ahiahonu PWK, 2005. Metabolism and detoxification of phytoalexins and analogs by

phytopathogenic fungi. Phytochemistry 66, 391–411.Pedras MSC, Yaya EE, Glawischnig E, 2011. The phytoalexins from cultivated and wild crucifers: chemistry

and biology. Natural Product Reports 28, 1381–1405.Peng M, Kuc J, 1992. Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and

on tobacco leaf disks. Phytopathology 82, 696–699.Perazzolli M, Moretto M, Fontana P, Ferrarini A, Velasco R, Moser C, Delledonne M, Pertot I, 2012. Downy

mildew resistance induced by Trichoderma harzianum T39 in susceptible grapevines partially mimics tran-scriptional changes of resistant genotypes. BMC Genomics 13, 660.

Petrov VD, Van Breusegem F, 2012. Hydrogen peroxide – a central hub for information flow in plant cells.AoB Plants 2012, pls014.

Polesani M, Bortesi L, Ferrarini A, Zamboni A, Fasoli M, Zadra C, Lovato A, Pezzotti M, Delledonne M,Polverari A, 2010. General and species-specific transcriptional responses to downy mildew infection in asusceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species. BMC Genomics 11, 117.

Qiang X, Zechmann B, Reitz MU, Kogel K-H, Schäfer P, 2012. The mutualistic fungus Piriformospora indicacolonizes Arabidopsis roots by inducing an endoplasmic reticulum stress–triggered caspase-dependent celldeath. Plant Cell 24, 794–809.

Qualley AV, Widhalm JR, Adebesin F, Kish CM, Dudareva N, 2012. Completion of the core β-oxidative path-way of benzoic acid biosynthesis in plants. Proceedings of the National Academy of Science USA 109,16383–16388.

Rancé I, Fournier J, Esquerré-Tugayé M-T, 1998. The incompatible interaction between Phytophthora parasit-ica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenasesequences. Proceedings of the National Academy of Sciences USA 95, 6554–6559.



Reimann S, Deising HB, 2005. Inhibition of efflux transporter-mediated fungicide resistance in Pyrenophoratritici-repentis by a derivative of 4′-hydroxyflavone and enhancement of fungicide activity. Applied andEnvironmental Microbiology 71, 3269–3275.

Rey P, Benhamou N, Tirilly Y, 1996. Ultrastructural and cytochemical studies of cucumber roots infected bytwo Pythium species with different modes of pathogenicity. Physiological and Molecular Plant Pathology49, 213–231.

Ride JP, 1983. Cell walls and other structural barriers in defence. In: Callow JA, ed. Biochemical Plant Pathol-ogy. Chichester: John Wiley & Sons, pp. 215–236.

Romero-Puertas MC, Laxa M, Mattè A, Zaninotto F, Finkemeier I, Jones AME, Perazzolli M, Vandelle E,Dietz K-J, Delledonne M, 2007. S-nitrosylation of peroxiredoxin promotes peroxynitrite-mediated tyrosinenitration. Plant Cell 19, 4120–4130.

Rommens CM, 2010. Barriers and paths to market for genetically engineered crops. Plant Biotechnology Jour-nal 8, 101–111.

Rustérucci C, Montillet J-L, Agnel J-P, Battesti C, Alonso B, Knoll A, Bessoule J-J, Etienne P, Suty L, BleinJ-P, Triantaphylidès C, 1999. Involvement of lipoxygenase-dependent production of fatty acid hydroperox-ides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves. Journalof Biological Chemistry 274, 36446–36455.

Samara C, Syntichaki P, Tavernarakis N, 2007. Autophagy is required for necrotic cell death in Caenorhabditiselegans. Cell Death and Differentiation 15, 105–112.

Schäfer W, Straney D, Ciuffetti L, Van Etten HD, Yoder OC, 1989. One enzyme makes a fungal pathogen, butnot a saprophyte, virulent on a new host plant. Science, 246, 247–249.

Schmelz EA, Kaplan F, Huffaker A, Dafoe NJ, Vaughan MM, Ni X, Rocca JR, Alborn HT, Teal PE, 2011.Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize. Proceedings of the NationalAcademy of Sciences USA 108, 5455–5460.

Schmelzer E, 2002. Cell polarization, a crucial process in fungal defence. Trends in Plant Science 7,411–415.

Schoonbeek H, Del Sorbo G, De Waard MA, 2001. The ABC transporter BcatrB affects the sensitivity ofBotrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil. Molecular Plant-Microbe Inter-actions 14, 562–571.

Schuhegger R, Rauhut T, Glawischnig E, 2007. Regulatory variability of camalexin biosynthesis. Journal ofPlant Physiology 164, 636–644.

Schutz I, Gus-Mayer S, Schmelzer E, 2006. Profilin and Rop GTPases are localized at infection sites of plantcells. Protoplasma 227, 229–235.

Schweichel JU, Merker HJ, 1973. The morphology of various types of cell death in prenatal tissues. Teratology7, 253–266.

Sels J, Mathys J, De Coninck BMA, Cammue BPA, De Bolle MFC, 2008. Plant pathogenesis-related (PR)proteins: A focus on PR peptides. Plant Physiology and Biochemistry 46, 941–950.

Seregélyes C, Igamberdiev AU, Maassen A, Hennig J, Dudits D, Hill RD, 2004. NO-degradation by alfalfa class1 hemoglobin (Mhb1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants.FEBS Letters 571, 61–66.

Shepherd DN, Martin DP, Thomson JA, 2009. Transgenic strategies for developing crops resistant to gemi-niviruses. Plant Science 176, 1–11.

Shimada C, Lipka V, O’Connel R, Okuno T, Schulze-Lefert P, Takano Y, 2006. Nonhost resistance inArabidopsis-Colletotrichum interactions acts at the cell periphery and requires actin filament function.Molecular Plant-Microbe Interactions 19, 270–279.

Simon C, Langlois-Meurinne M, Bellvert F, Garmier M, Didierlaurent L, Massoud K, Chaouch S, Marie A,Bodo B, Kauffmann S, Noctor G, Saindrenan P, 2010. The differential spatial distribution of secondarymetabolites in Arabidopsis leaves reacting hypersensitively to Pseudomonas syringae pv. tomato is depen-dent on the oxidative burst. Journal of Experimental Botany 61, 3355–3370.

Skalamera D, Heath MC, 1996. Cellular mechanisms of callose deposition in response to fungal infection orchemical damage. Canadian Journal of Botany 74, 1236–1242.

Smart MG, Aist JR, Israel HW, 1986. Structure and function of wall appositions. 1. General histochemistry ofpapillae in barley coleoptiles attacked by Erysiphe graminis f. sp. hordei. Canadian Journal of Botany 64,793–801.

Snyder BA, Nicholson RL, 1990. Synthesis of phytoalexins in sorghum as a site-specific response to fungalingress. Science (New York, NY) 248, 1637–1639.



Sobolev VS, 2008. Localized production of phytoalexins by peanut (Arachis hypogaea) kernels in response toinvasion by Aspergillus species. Journal of Agricultural and Food Chemistry 56, 1949–1954.

Sobolev VS, Neff SA, Gloer JB, 2009. New stilbenoids from peanut (Arachis hypogaea) seeds challenged byan Aspergillus caelatus strain. Journal of Agricultural and Food Chemistry 57, 62–68.

Sobolev VS, Khan SI, Tabanca N, Wedge DE, Manly SP, Cutler SJ, Coy MR, Becnel JJ, Neff SA, GloerJB, 2011. Biological activity of peanut (Arachis hypogaea) phytoalexins and selected natural and syntheticstilbenoids. Journal of Agricultural and Food Chemistry 59, 1673–1682.

Soylu EM, Soylu S, Mansfield JW, 2004. Ultrastructural characterisation of pathogen development and hostresponses during compatible and incompatible interactions between Arabidopsis thaliana and Peronosporaparasitica. Physiological and Molecular Plant Pathology 65, 67–78.

Stackman CE, 1915. Relation between Puccinia graminis and plants highly resistant to its attack. Journal ofAgricultural Research 4, 193–199.

Stanjek V, Piel J, Boland W, 1999. Biosynthesis of furanocoumarins: mevalonate-independent prenylation ofumbelliferone in Apium graveolens (Apiaceae). Phytochemistry 50, 1141–1146.

Stefanato FL, Abou-Mansour E, Buchala A, Kretschmer M, Mosbach A, Hahn M, Bochet CG, Metraux J-P,Schoonbeek HJ, 2009. The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a viru-lence factor on Arabidopsis thaliana. The Plant Journal 58, 499–510.

Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K, 2000. Synergy in a medicinal plant: antimicrobialaction of berberine potentiated by 5’-methoxyhydnocarpin, a multidrug pump inhibitor. Proceedings of theNational Academy of Sciences USA 97, 1433–1437.

Stoitsova SO, Braun Y, Ullrich MS, Weingart H, 2008. Characterization of the RND-type multidrug effluxpump MexAB-OprM of the plant pathogen Pseudomonas syringae. Applied and Environmental Microbiol-ogy 74, 3387–3393.

Stone BA, Clarke AE, 1992. Chemistry and Biology of (1-3)-β-D-glucan. Victoria, Australia: La TrobeUniversity Press.

Subramanian S, Graham MY, Yu O, Graham TL, 2005. RNA interference of soybean isoflavone synthase genesleads to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthorasojae. Plant Physiology 137, 1345–1353.

Sweetlove LJ, Fernie AR, 2013. The spatial organization of metabolism within the plant cell. Annual Reviewof Plant Biology 64, 723–746.

Tegos G, Stermitz FR, Lomovskaya O, Lewis K, 2002. Multidrug pump inhibitors uncover remarkable activityof plant antimicrobials. Antimicrobial Agents and Chemotherapy 46, 3133–3141.

Thordal-Christensen H, 2003. Fresh insights into processes of nonhost resistance. Current Opinion in PlantBiology 6, 351–357.

Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB, 1997. Subcellular localization of H2O2 in plants. H2O2

accumulation in papillae and hypersensitive response during the barley powdery mildew interaction. ThePlant Journal 11, 1187–1194.

Ton J, Jakab G, Toquin V, Flors V, Iavicoli A, Maeder MN, Métraux J-P, Mauch-Mani B, 2005. Dissectingthe β-aminobutyric acid-induced priming phenomenon in Arabidopsis. Plant Cell 17, 987–999.

Ton J, Flors V, Mauch-Mani B, 2009. The multifaceted role of ABA in disease resistance. Trends in PlantScience 14, 310–317.

Torres MA, Dangl JL, Jones JDG, 2002. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are requiredfor accumulation of reactive oxygen intermediates in the plant defense response. Proceedings of the NationalAcademy of Sciences USA 99, 517–522.

Umezawa T, 2003. Diversity in lignan biosynthesis. Phytochemistry Reviews 2, 371–390.Urban M, Bhargava T, Hamer JE, 1999. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast

disease. Embo Journal 18, 512–521.Uren AG, O’Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM, 2000. Identification of

paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key rolein MALT Lymphoma. Molecular Cell 6, 961–967.

van Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, Jones AM,Kawai-Yamada M, Lam E, Mundy J, Mur LAJ, Petersen M, Smertenko A, Taliansky M, Van BreusegemF, Wolpert T, Woltering E, Zhivotovsky B, Bozhkov PV, 2011. Morphological classification of plant celldeaths. Cell Death and Differentiation 18, 1241–1246.

Van Loon LC, Van Strien EA, 1999. The families of pathogenesis-related proteins, their activities, and com-parative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology 55, 85–97.



Van Loon LC, Rep M, Pieterse CMJ, 2006. Significance of inducible defense-related proteins in infected plants.Annual Review of Phytopathology 44, 135–162.

Vance CP, Kirk TK, Sherwood RT, 1980. Lignification as a mechanism of disease resistance. Annual Reviewof Phytopathology 18, 259–288.

Vandelle E, Delledonne M, 2011. Peroxynitrite formation and function in plants. Plant Science 181, 534–539.

VanEtten HD, Mansfield JW, Bailey JA, Farmer EE, 1994. Two classes of plant antibiotics - phytoalexins versusphytoanticipins. Plant Cell 6, 1191–1192.

VanEtten HD, Temporini E, Wasmann CC, 2001. Phytoalexin (and phytoanticipin) tolerance as a virulencetrait: why is it not required by all pathogens? Physiological and Molecular Plant Pathology 59, 83–93.

Vargas P, Felipe A, Michán C, Gallegos M-T, 2011. Induction of Pseudomonas syringae pv. tomato DC3000MexAB-OprM multidrug efflux pump by flavonoids is mediated by the repressor PmeR. MolecularPlant-Microbe Interactions 24, 1207–1219.

von Röpenack E, Parr A, Schulze-Lefert P, 1998. Structural analyses and dynamics of soluble and cellwall-bound phenolics in a broad spectrum resistance to the powdery mildew fungus in barley. Journal ofBiological Chemistry 273, 9013–9022.

Vorwerk S, Vorwerk S, Somerville S. 2004. The role of plant cell wall polysaccharide composition in diseaseresistance. Trends in Plant Science 9, 203–209.

Walley JW, Dehesh K, 2010. Molecular mechanisms regulating rapid stress signaling networks in Arabidopsis.Journal of Integrative Plant Biology 52, 354–359.

Wally O, Jayaraj J, Punja ZK, 2009. Broad-spectrum disease resistance to necrotrophic and biotrophicpathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene. Planta 231,131–141.

Wang X, 2011. Structure, function, and engineering of enzymes in isoflavonoid biosynthesis. Functional andIntegrative Genomics 11, 13–22.

Wasmann CC, VanEtten HD, 1996. Transformation-mediated chromosome loss and disruption of a gene forpisatin demethylase decrease the virulence of Nectria haematococca on pea. Molecular Plant-Microbe Inter-actions 9, 793–803.

Winkel BSJ, 2004. Metabolic channeling in plants. Annual Review of Plant Biology 55, 85–107.

Wolf S, Hematy K, Hofte H, 2012. Growth Control and Cell Wall Signaling in Plants. Annual Review of PlantBiology, 63, 381–407.

Wood RKS, 1967. Physiological Plant Pathology. Oxford: Blackwell Scientific.

Wu Q, VanEtten HD, 2004. Introduction of plant and fungal genes into pea (Pisum sativum L.) hairyroots reduces their ability to produce pisatin and affects their response to a fungal pathogen. MolecularPlant-Microbe Interactions 17, 798–804.

Wyllie AH, Golstein P, 2001. More than one way to go. Proceedings of the National Academy of Sciences USA98, 11–13.

Xu G, Li S, Xie K, Zhang Q, Wang Y, Tang Y, Liu D, Hong Y, He C, Liu Y, 2012. Plant ERD2-like proteinsfunction as endoplasmic reticulum luminal protein receptors and participate in programmed cell death duringinnate immunity. The Plant Journal 72, 57–69.

Yamane H, 2013. Biosynthesis of phytoalexins and regulatory mechanisms of it in rice. Bioscience Biotech-nology and Biochemistry 77, 1141–1148.

Yoshikawa M, Yamauchi K, Masago H, 1978. Glyceollin: its rôle in restricting fungal growth in resistantsoybean hypocotyls infected with Phytophthora megasperma var. sojae. Physiological Plant Pathology 12,73–82.

Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JDG, Doke N, 2003. Nico-tiana benthamiana gp91phox homologs NBRBOHA and NBRBOHB participate in H2O2 accumulationand resistance to Phytophthora infestans. The Plant Cell 15, 706–718.

Yu IC, Parker J, Bent AF, 1998. Gene-for-gene disease resistance without the hypersensitive response in Ara-bidopsis dnd1 mutant. Proceedings of the National Academy of Sciences USA 95, 7819–7824.

Yun BW, Feechan A, Yin M, Saidi NBB, Le Bihan T, Yu M, Moore JW, Kang J-G, Kwon E, Spoel SH, PallasJA, Loake GJ, 2011. S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature, 478,264–268.

Zeyen R, Carver TLW, Lyngkjaer MF, 2002. Epidermal cell papillae. In: Belanger RR, Bushnell WR, Dik AJ,Carver TLW, eds. The Powdery Mildews: A Comprehensive Treatise. St. Paul, MN: American Phytopatho-logical Society, pp. 107–124.



Zhang H, Fang Q, Zhang Z, Wang Y, Zheng X, 2009. The role of respiratory burst oxidase homologues inelicitor-induced stomatal closure and hypersensitive response in Nicotiana benthamiana. Journal of Exper-imental Botany 60, 3109–3122.

Zhang Z, Feechan A, Pedersen C, Newman M-A, Qiu J-l, Olesen KL, Thordal-Christensen H, 2007. ASNARE-protein has opposing functions in penetration resistance and defence signalling pathways. The PlantJournal 49, 302–312.

Zhao J, Davis LC, Verpoorte R, 2005. Elicitor signal transduction leading to production of plant secondarymetabolites. Biotechnology Advances 23, 283–333.

Zhao Q, Dixon RA, 2011. Transcriptional networks for lignin biosynthesis: more complex than we thought?Trends in Plant Science 16, 227–233.

Zhou H, Lin J, Johnson A, Morgan RL, Zhong W, Ma W, 2011. Pseudomonas syringae type III effector HopZ1targets a host enzyme to suppress isoflavone biosynthesis and promote infection in soybean. Cell Host andMicrobe 9, 177–186.

Zhu YJ, Agbayani R, Jackson MC, Tang CS, Moore PH, 2004. Expression of the grapevine stilbene synthasegene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora.Planta 220, 241–250.

Zimmerli L, Jakab C, Metraux JP, Mauch-Mani B, 2000. Potentiation of pathogen-specific defense mecha-nisms in Arabidopsis by β-aminobutyric acid. Proceedings of the National Academy of Sciences USA 97,12920–12925.

Zottini M, Formentin E, Scattolin M, Carimi F, Lo Schiavo F, Terzi M, 2002. Nitric oxide affects plant mito-chondrial functionality in vivo. FEBS Letters 515, 75–78.

Zubieta C, Kota P, Ferrer J-L, Dixon RA, Noel JP, 2002. Structural basis for the modulation of lignin monomermethylation by caffeic acid/5-hydroxyferulic acid 3/5-o-methyltransferase. The Plant Cell 14, 1265–1277.

Zuluaga AP, Vega-Arreguín JC, Fry WE, 2013. Transcriptome profile of acibenzolar-S-methyl-induced genesin tomato suggests a complex polygenic effect on resistance to Phytophthora infestans. Physiological andMolecular Plant Pathology 81, 97–106.

Documents

Induced Resistance for Plant Defense (Walters/Induced Resistance for Plant Defense) || Mechanisms of Defence to Pathogens: Biochemistry and Physiology