Plant Science 240 (2015) 9097

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Review article

Immunity to plant pathogens and iron homeostasis

Aude Aznar a , Nicolas W.G. Chen b , Sebastien Thomine c , Alia Dellagi a,
a

Institut Jean-Pierre Bourgin, UMR INRA-AgroParisTech 1318, ERL CNRS 3559, Saclay Plant Sciences, RD10, F-78026 Versailles, France
Institut de Recherche en Horticulture et Semences, UMR1345 INRA-AgroCampus-Ouest, F-49045 Angers, France
c
Institute for Integrative Biology of the Cell (I2BC), CNRS, Gif-sur-Yvette 91198, France
b

a r t i c l e

i n f o

Article history:
Received 31 May 2015
Received in revised form 27 August 2015
Accepted 28 August 2015
Available online 29 August 2015
Keywords:
Plant immunity
Plant defense
Iron
Pathogen
Reactive oxygen species
Phenolics
Siderophores

a b s t r a c t
Iron is essential for metabolic processes in most living organisms. Pathogens and their hosts often compete
for the acquisition of this nutrient. However, iron can catalyze the formation of deleterious reactive oxygen species. Hosts may use iron to increase local oxidative stress in defense responses against pathogens.
Due to this duality, iron plays a complex role in plant-pathogen interactions. Plant defenses against
pathogens and plant response to iron deciency share several features, such as secretion of phenolic
compounds, and use common hormone signaling pathways. Moreover, ne tuning of iron localization
during infection involves genes coding iron transport and iron storage proteins, which have been shown
to contribute to immunity. The inuence of the plant iron status on the outcome of a given pathogen
attack is strongly dependent on the nature of the pathogen infection strategy and on the host species.
Microbial siderophores emerged as important factors as they have the ability to trigger plant defense
responses. Depending on the plant species, siderophore perception can be mediated by their strong iron
scavenging capacity or possibly via specic recognition as pathogen associated molecular patterns. This
review highlights that iron has a key role in several plant-pathogen interactions by modulating immunity.
2015 Published by Elsevier Ireland Ltd.

Contents
1.
2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Interplay between iron homeostasis and immunity responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.1.
Plant iron deciency triggers accumulation of antimicrobial phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.2.
Iron homeostasis genes are involved in immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
2.3.
Iron is involved in ROS accumulation in plant-pathogen interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
2.4.
Defense signals are involved in iron homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
2.5.
Microbial siderophores manipulate plant iron homeostasis and immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Conclusions and open questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

1. Introduction
Crop production is threatened by biotic stresses. The use of
pesticides to limit diseases must be reduced because of their detrimental effects on environment and human health. Thus, novel
sustainable plant protection strategies must be developed. For
this, a better understanding of the mechanisms underlying plantpathogen interactions is necessary.

Corresponding author: Institut Jean-Pierre Bourgin, UMR INRA-AgroParisTech

1318, INRA Center de Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles
Cedex, France.
E-mail address: dellagi@agroparistech.fr (A. Dellagi).
http://dx.doi.org/10.1016/j.plantsci.2015.08.022
0168-9452/ 2015 Published by Elsevier Ireland Ltd.

Many biotic stresses are caused by microbial pathogens such as

fungi or bacteria that invade and develop on plant tissues using different infection strategies. Some pathogens are obligate parasites
with a biotrophic development where plant cells are maintained
alive (e.g. the downy mildew agent Hyaloperonospora arabidopsidis, [1]). Others use a necrotrophic strategy in which pathogen
multiplies in dead plant tissue (e.g. the gray mold agent Botrytis
cinerea [2]). Interestingly, some pathogens start their infection process with a biotrophic phase and then shift to a necrotrophic phase.
They are referred to as hemibiotrophic (e.g. the anthracnose agent
Colletotrichum graminicola [3]).
Plants have evolved sophisticated defense mechanisms that
allow detection of environmental cues and translate them into

A. Aznar et al. / Plant Science 240 (2015) 9097

physiological responses. In the last few years, major advances have

been achieved toward a better understanding of pathogen perception by plants and the consequent activation of immunity processes
[4]. The plants innate immune system detects pathogen attack and
activates complex signaling networks leading to induced defenses
that confer a more tolerant state. Activation of this signaling
network is achieved via receptors transmitting signals to downstream components that trigger formation of physical and chemical
defenses. Induced defenses require phosphorylation events and
defense hormone signaling leading to the accumulation of reactive
oxygen species (ROS), cell wall strengthening, callose deposition
and expression of pathogenesis related (PR) genes [5].
Plants produce pattern recognition receptors [6] that recognize
molecular motifs conserved amongst a microbial taxonomic group,
such as chitin which is a common component of the cell wall in
most fungi [7] or lipopolysaccharide which is common component
of the envelope of all gram negative bacteria [8]. These conserved
motifs are called microbial associated molecular patterns (MAMPs).
Recognition of MAMPs by pattern recognition receptors leads to the
activation of a basal defense response process called pattern triggered immunity [9,10]. During plant infection, pathogens secrete
different types of molecules with various effects on plants [11].
For instance, several pathogens secrete plant cell wall degrading
enzymes or toxins that are secondary metabolites that disrupt plant
metabolism. Many pathogens also secrete and inject proteins called
effectors into plant cells that favor infection and plant invasion
by suppressing pattern triggered immunity [12]. Within a plant
species, some genotypes are able to specically detect an effector
via a resistance (R) protein. The recognition of an effector by an R
protein leads to a form of localized cell death called hypersensitive response (HR). The HR zone restricts the pathogen to the site
of attempted infection and prevents spread of the disease.
The molecules recognized by plant cells in the context of
pathogen interaction, such as MAMPs or effectors, are referred to
as elicitors. In some cases, detection of elicitors directly activates
defense responses (PR gene expression, ROS accumulation. . .). In
other cases, elicitor recognition does not lead to the immediate
induction of defense responses, but instead allows the plant to activate responses more rapidly and/or more strongly in response to
future pathogen attacks. The latter process is called priming [13].
Plant immunity processes are inuenced by nutrition and by
the plant metabolic status [14]. For instance, accumulation of callose in Arabidopsis thaliana in response to chitosan depends on
the presence of sucrose and vitamins in the growth medium [15].
Likewise, plant nitrate nutrition affects HR in tobacco plants [16].
Several reports showed the important role of iron in plant pathogen
virulence pointing to a hypothetical function for iron in the host
immune system (for review: [17]).
Iron is an essential element for nearly all life forms. It is required
as a co-factor for the activity of many proteins involved in essential
cellular processes, such as the electron transfer chain in respiration,
photosynthesis, DNA synthesis and detoxication of ROS. Under
aerobic conditions at high pH, iron is present as insoluble ferric
(FeIII) hydroxide complexes making it poorly available for living
organisms. Under reductive conditions, iron is found under the soluble ferrous form (FeII). FeII is however potentially toxic to living
cells as, in the presence of hydrogen peroxide, free Fe2+ catalyses
the Fenton reaction, leading to the formation of hydroxyl radicals
that cause protein damage, DNA breaks and lipid peroxidation [18].
Therefore iron acquisition, utilization and storage are tightly controlled at the cell and tissue levels to provide sufcient amounts for
metabolism while preventing accumulation of ferrous iron.
Iron is taken up from the soil through the roots then it is
transported into the xylem as a complex with organic acids such
as citrate. In the phloem Fe is often complexed with the nonproteinaceaous amino-acid nicotianamine. Many genes involved in

91

Fe trafcking between the apoplast and the cells and between the
cytoplasm and organelles have been cloned and characterized (for
review: [19,20]). Dicots such as A. thaliana and non-graminaceous
monocots use a reductive Fe uptake mechanism described as Strategy I (Fig. 1). In these plants, secretion of phenolics and acidication
of the soil by the action of H+ -ATPases solubilize ferric iron complexes in the rhizosphere. Subsequently, Fe(III) is reduced by the
ferric chelate reductase FRO2, prior to its transport across the
plasma membrane of root epidermal cells via the Fe transporter
IRT1. Storage and buffering in dedicated cell compartments including the apoplast and organelles (vacuole, plastids) allow protection
against Fe toxicity. High Fe concentrations have been observed in
plant nuclei [21] but the role of nuclei in Fe storage as well as the
role of Fe in nuclei remain to be elucidated. In plastids, ferritins
encoded by FER genes allow Fe storage under a safe non-reactive
but readily available from [22]. Iron may also be stored in the vacuole and mobilization from this organelle is mediated by NRAMP3
and NRAMP4 proteins. The central regulator of the Fe deciency
response in A. thaliana roots is the basic helix-loop-helix (bHLH)
transcription factor FIT [23]. Other transcription factors of the bHLH
family (bHLH100, bHLH101, bHLH38, bHLH39, PYE) contribute to
a wide network that ne tunes iron uptake upon deciency in
roots and shoots. The transcription factors MYB10 and MYB72 were
found to be required for A. thaliana adaptation to low Fe indicating
their possible role in the overall response to iron deciency [24].
Graminaceae plants use Strategy II to acquire iron from soil
(Fig. 1). Their roots secrete FeIII chelators of the mugineic acid family called phytosiderophores (PS). The secretion of PS in rice and
barley is achieved via the transporter TOM1 [25]. Following iron
complexation by PS in the rhizosphere, graminaceous plants take
up Fe-PS complexes through the action of transporters of the Yellow Stripe 1 (YS1) family [26]. The regulation of the response to iron
deciency in rice is mediated by the transcription factors IDEF1,
IDEF2, IRO2 and IRO3. IDEF1 belongs to the plant specic family
VP1/ABI3. As IDEF1 transcription is not regulated by iron and IDEF1
protein binds Fe and Zn, IDEF1 was proposed to act as a primary
Fe sensor [23]. IDEF2 belongs to the NAC family and IRO2 and IRO3
belong to the bHLH family. They bind distinct specic cis-regulatory
sequences identied in the promoter of genes responsive to iron
deciency [23].
The most widespread mechanism of iron uptake by microbes is
based on the secretion of low molecular weight (less than 1 kDa)
siderophores with high afnity for FeIII. As in Strategy II plants,
siderophores are secreted under iron deciency to solubilize and
scavenge FeIII and are then taken up by the microbial cell as ferric
siderophore complexes. Siderophores are produced by virtually all
micro-organisms but display a range of chemical structures that
differ in their afnities for iron. (for review see [27]).
The sophisticated strategies developed by plants and microbes
to control their own Fe homeostasis are in conict during plantpathogen interactions. Here, the role of iron in plant immunity is
discussed with a special emphasis on recent reports that shed light
on mechanistic links between plant iron homeostasis and different
defense processes.

2. Interplay between iron homeostasis and immunity

responses
2.1. Plant iron deciency triggers accumulation of antimicrobial
phenolics
Phenylpropanoids are produced by all plants. They derive from
the amino-acid l-phenyl-alanine (Phe). The rst step of the phenylpropanoid pathway is the deamination of Phe via the enzyme
phenylalanine amonia-lyase (PAL) [28]. A wide array of secondary

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A. Aznar et al. / Plant Science 240 (2015) 9097

Fig.1. Strategy I and Strategy II: the two different models of Angiosperm iron acquisition mechanisms.
Strategy I describes the dicots iron acquisition from rhizosphere and strategy II describe the Graminaceae monocots iron aquisition from rhizosphere.
In Strategy I, secretion of phenolics and acidication of the soil by the action of H+ -ATPases solubilize ferric iron complexes. The ferric chelate reductase FRO2 reduces FeIII
into FeII, and nally FeII is transported across the plasma membrane of root epidermal cells via the iron transporter IRT1.
In Strategy II, Fe chelators from the mugineic acid family called phytosiderophores (PS) are secreted at the root level by the transporter TOM1 in rice and barley. In the
rhizosphere, FeIII is chelated by PS creating a Fe-PS complex transported across the plasma membrane of root epidermal cells via transporters of the Yellow Stripe 1 family
(YS1/YSL).

compounds with different biological activities including pharmaceutical compounds is produced through this pathway [28,29].
Phenylpropanoids include avonoids, coumarins, anthocyanins
and lignin. This pathway is also a route for the biosynthesis of many
antimicrobial compounds, such as pterocarpan or resveratrol, and
the well-known defence hormone salicylic acid that accumulate
in plants in response to pathogen infection [30]. It has been known
since the 1980s that plant roots secrete phenolic compounds under
iron deciency [31]. The genes, proteins and compounds involved
in the phenylpropanoid pathway are often up-regulated in Fe decient plants. For example, a phe-ammonia-lyase, two coumarate:
CoA ligases (4CL1 and 4CL2), and an oxido-reductase involved in
coumarin synthesis (F6 H1) are up-regulated under Fe deciency in
A. thaliana [32]. The F6 H1 gene encodes an enzyme required for the
hydroxylation of feruloyl-CoA, a prerequisite for further formation
of coumarins like scopolin, scopoletin and hydroxyl-feruloyl-CoA.
A T-DNA f6 h1 insertion mutant was identied in a screen looking for A. thaliana mutants unable to grow on alkaline soils, where
Fe is poorly available [33]. In this f6 h1 mutant, defective growth
under Fe decient conditions is associated with inability to secrete
several coumarins including scopolin, scopoletin, esculin, esculetin
and fraxetin in the rhizosphere [33]. PDR9 encodes an ABC transporter involved in secretion of several phenylpropanoids including
scopoletin into the rhizosphere; a pdr9 mutant grows very poorly
under Fe decient conditions [34]. Furthermore, the up-regulation
of F6 H1 and AtPDR9 genes under Fe deciency is dependent on
the master regulator of iron deciency response, FIT. Thus, production and secretion of scopoletin is controlled by the plant Fe
specic signalling network. Interestingly, scopoletin was characterized as a phytoalexin with antifungal activity [35]. Scopoletin
has a direct inhibition potential against the fungus A. alternata
and tobacco plants silenced for the enzyme F6 H1 are more susceptible to this pathogen [36]. In rice, Fe deciency triggers the
secretion of several phenolics including protocatechuic acid which
can derive from shikimate (a precursor of Phe) [37]. Indeed, protocatechuic acid binds iron and is required for solubilization of iron

precipitated in the root apoplast [29,37,38]. Accordingly, the rice

pez1 mutant affected in the secretion of protocatechuic acid into
the xylem contains higher iron concentrations in roots [39]. Interestingly, protocatechuic acid also has antimicrobial activity and
confers resistance to the fungal pathogen Colletotrichum circinians
in onion [11]. These data show that several phenolic compounds
have dual functions in plants: they improve iron nutrition and protect the plant against pathogens.
The accumulation of such compounds is generally interpreted
as a mechanism by which plants solubilize cell wall iron or external sources of iron when this element is not available. Indeed,
plant cell walls contain high amounts of iron that can be mobilized
by these phenolic compounds [19]. Many molecules with strong
antimicrobial activity have at the same time strong iron solubilizing
and binding properties. Coupling these properties likely provides
a tness advantage to the plant. The antimicrobial effect could be
directly due to the iron scavenging property of these molecules
as described in animals for a long time as nutritional immunity
were Fe scavenging by lactoferrin inhibits microbial proliferation
[40]. Iron withdrawal via scavengers such as transferrin or lipocalin
renders this element poorly available to pathogens [41]. However,
in most cases, the Fe binding properties of phenolic compounds
secreted by plants under Fe deciency remain to be established.
Alternatively, these molecules may intrinsically possess two separate activities, Fe binding and antimicrobial effect. It is conceivable
that the production of molecules allowing both mobilization of Fe
stores and protection against invaders when a plant is weakened
by Fe deciency have been positively selected through evolution.
The fact that Fe starvation leads to the accumulation of antimicrobial compounds suggests that Fe decient plants may be
expected to be more resistant to pathogens than non-decient
plants. Several reports support this hypothesis. For example, Fe
starved A. thaliana plants are more resistant to the bacterial
necrotrophic pathogen Dickeya dadantii [42]. Fe deciency also
conferred increased tolerance to the fungal necrotroph B. cinerea

A. Aznar et al. / Plant Science 240 (2015) 9097

[42,43]. Whether phenolic compound production is involved in the

increased resistance remains to be demonstrated.
Iron starvation responses may be triggered locally by strong
Fe scavenging molecules such as siderophores, which are
secreted during infection. Several reports have shown that
siderophores or chemicals with strong iron chelating capacity trigger immune responses. For instance, deferrioxamine
chrysobactin, or the synthetic iron chelator ethylenediamineN,N -bis(2-hydroxyphenylacetic) acid (EDDHA) trigger defence
responses in A. thaliana [44,45]. Conversely, Koen et al. [43] found
that -amino butyric acid (BABA) a molecule known to prime
plant immunity was able to bind Fe in vitro and to trigger physiological responses similar to Fe deciency responses including
up-regulation of Fe deciency genes and appearance of leaf interveinal chlorosis. Consistently, similar metabolomics proles are
observed in BABA-treated and Fe-starved plants, with strong accumulation of anthocyanins and coumarins [43].
Altogether, these data conrm the strong connection between
immunity and Fe deciency responses in plants. Plants could have
evolved a signalling system that interprets Fe depletion caused by
Fe scavenging by microbial siderophores as a signal for pathogen
infection. Sensing Fe withdrawal might thus have been evolutionarily linked to immunity.
2.2. Iron homeostasis genes are involved in immunity
Several genes involved in plant Fe homeostasis are up-regulated
upon biotic stresses suggesting that infection disturbs Fe homeostasis [4650]. The role of some Fe homeostasis genes in plant
resistance to pathogens has been studied using either T-DNA
insertion mutants or over-expressing lines (Table 1). For instance,
infection by D. dadantii triggered up-regulation of several genes
involved in Fe transport including AtNRAMP3, IRT1, FRO2 tting
into the hypothesis that infection causes an Fe deciency-like signal [47]. These genes were shown to be involved in tolerance to D.
dadantii in A. thaliana. [44,47].
Over-expression of an alfalfa ferritin gene in tobacco conferred resistance to the tobacco necrosis virus and to the fungal
necrotrophic pathogens A. alternata and B. cinerea [51]. In addition to tolerance to pathogens, the transformed plants were more
tolerant to oxidative stress caused by paraquat. It is unlikely that
plant ferritin can withhold Fe from pathogens because microbial
siderophores have an extremely high afnity for Fe and are generally able to cause Fe release from ferritin in animal systems
[52]. Thus, the role of ferritin in plant defense is more likely to
be a protection against oxidative stress mediated cell death. In a
recent report, Aznar et al. [53] showed that a strong depletion of
Fe occurs in leaf tissues colonized by D. dadantii bacteria whereas
ahead of the colonized zones, healthy plant cells still contained
Fe and accumulated ferritin. These data suggest that during infection, it is necessary to buffer Fe released from dying plant cells to
limit Fe mediated ROS accumulation that could be deleterious to
healthy neighboring plant cells. Consistently, the A. thaliana AtFER1
insertion mutant showed increased susceptibility to the bacterial
pathogen D. dadantii [46]. Together these data show that ferritin
contributes to defense against several necrotrophic pathogens and
that this is strongly linked to ferritin antioxidant function. The role
of ferritin in plant interactions with biotrophic fungi remains to be
investigated.
Proteins involved in metal transport across the plant vacuolar
membrane share similarities with NRAMP described in animals.
In mouse, NRAMP1 is involved in metal efux from endosomes
containing intracellular pathogens, thus depleting endosomes of
essential metals [54]. Such proteins have the same role in Fe homeostasis in several plants. Tolerance to the bacterial pathogen D.
dadantii was shown to involve AtNRAMP3 and to a lesser extend

93

AtNRAMP4 [47]. Transcriptional AtNRAMP3 gene expression is upregulated probably as a result of an Fe deciency like signal
triggered by infection. The authors found that AtNRAMP3 is involved
in amplication of ROS formation and in ferritin accumulation in
response to D. dadantii. Thus, it could be hypothesized that the
role of AtNRAMP3 in defense is related to Fe efux from the vacuole that results in ROS formation and ferritin accumulation. In
rice, OsNRAMP6 was found to be regulated by the osa-miR7695
miRNAs in response to the fungal pathogen Magnaporthae oryzae
derived elicitors [55]. In rice overexpressing osa-miR7695, the lower
expression of OsNRAMP6 is associated with reduced symptoms
after inoculation with M. oryzae. Thus a tight regulation of plant
Fe homeostasis contributes to protection against pathogens.
Iron homeostasis may also contribute to the activation of immunity by benecial microbes to allow protection against pathogens.
Induced systemic resistance (ISR) is a well described process
of systemic immunity triggered by non pathogenic rhizobacteria [56]. Interestingly, myb72 A. thaliana mutants fail to establish
induced systemic resistance triggered by non-pathogenic rhizobacteria [57]. The transcription factor MYB72 was found to regulate
several Fe transporters during ISR [58]. Furthermore, the fact that
siderophores secreted by benecial bacteria can trigger ISR [56],
indicates that Fe has a crucial role in this process potentially via the
action MYB72. Since, the transcription factor MYB72 was described
as being involved in plant adaptation to low Fe [24], it would be
useful to study its role in the cross talk between Fe and immunity.
2.3. Iron is involved in ROS accumulation in plant-pathogen
interactions
Reactive oxygen species have pivotal roles in plant pathogeninteractions. They act as direct antimicrobial compounds and
contribute to the strengthening of cell walls by triggering cross
linking between hydroxy-proline rich proteins and callose apposition [59]. A strong oxidative burst is generally associated with
resistance, such as during hypersensitive response, for example.
Several mechanisms for ROS formation in response to pathogens
have been described. The NADPH-oxidase encoded by A. thaliana
AtRbohD has a crucial role in initial ROS production and resistance
to several pathogens [60]. Peroxidases are also involved in apoplastic ROS production in response to pathogens [61]. Although free Fe
is well known to catalyse ROS formation [18], little information is
available on the role of this element in ROS production in response
to pathogens. Oxidative stress may be more or less exacerbated by
available Fe depending on the plant Fe status. Consistent with this
idea, maize Fe nutrition has a strong impact on susceptibility to the
pathogenic hemibiotrophic fungus C. graminicola [62]. Iron starved
maize plants are more susceptible to infection, while adequate Fe
nutrition conferred a more tolerant state. Increased tolerance was
attributed to increased ROS production coinciding with Fe accumulation at infection sites during the early biotrophic phase. Iron
starved plants do not display these responses [62]. In agreement
with these ndings, microscopic observations using Prussian blue
staining showed that during early stages of barley infection by the
mildew agent Blumeria graminis, Fe is concentrated under the penetration sites [48]. The Fe spots co-localized with ROS accumulation
detected by 3-3 diaminobenzidine (DAB) staining. The Fe accumulation under fungal appressoria is mediated by vesicular trafcking
[48]. Moreover, Fe starved A. thaliana plants are also unable to produce ROS in response to D. dadantii compared to plants grown under
sufcient Fe conditions [42].
These data indicate that Fe has a role in ROS formation during plant pathogen interactions. However, the function of ROS may
depend on the infection strategy of the invader. Biotrophy might be
counteracted by Fe because it contributes to local ROS mediated cell
death, as presumably in the case for B. graminis/barley interaction

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A. Aznar et al. / Plant Science 240 (2015) 9097

Table 1
Plant genotypes with modied Fe homeostasis and their interaction with pathogens.
Plant species

Fe homeostasis gene

Mutation or over-expression

Phenotype

Reference

Tobacco
Tobacco
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Rice
Rice
Pear
Pear

Alfalfa ferritin
Human lactoferrin
AtFER1
NRAMP3-NRAMP4
IRT1
MYB72
YSL3
OsNRAMP6
OsNRAMP6
Pea ferritin
Bovine lactoferrin

Over-expression
Over-expression
T-DNA insertion mutant
T-DNA insertion double mutant
T-DNA insertion mutant
T-DNA insertion mutants
T-DNA insertion mutants
T-DNA insertion mutant
Over-expression
Over-expression
Over-expression

Resistance to B. cinerea, A. alternata, TNV and to oxidative stress

Resistance to Ralstonia solanacearum
Enhanced susceptibility to D. dadantii
Enhanced susceptibility to D. dadantii
Enhanced susceptibility to D. dadantii
Unable to mount induced systemic resistance
Enhanced susceptibility to P. syringae pv. tomato DC3000
Enhanced resistance to M. oryzae
Enhanced susceptibility to M. oryzae
No phenotype against Erwinia amylovora
Enhanced resistance E. amylovora

[51]
[91]
[46]
[47]
[44]
[57]
[77]
[55]
[55]
[92]
[93]

[48]. Conversely, Fe may favor necrotroph invasion by promoting

ROS mediated cell death, as in the case for B. cinerea, which requires
cell death to infect its host [63].
Iron deciency leads to the accumulation of antimicrobial
compounds such as phenylpropanoids and thus contributes to
immunity; but Fe may be needed at specic sites to trigger ROS and
hinder pathogen progression. This paradox is also well described in
animals where anemia or Fe overload may increase infectious diseases [41]. Depending on the infection strategy of the pathogen and
its intracellular or extracellular localization, the host Fe status may
have opposite consequences on immunity. Thus, not only Fe content must be considered in plant-pathogen interactions but also Fe
localization. In this context, using the new imaging techniques that
allow studying metal localization and valency at the cellular level
could be of great value [64].

2.4. Defense signals are involved in iron homeostasis

Ethylene, jasmonate and salicylic acid which are involved in
activation of immune responses, also modulate Fe homeostasis. Ethylene precursors up-regulate the expression of root genes
involved in Fe uptake in tomato, cucumber and A. thaliana while
inhibitors of ethylene repressed their expression [65,66]. Strikingly,
among the proteins interacting with FIT, two essential transcription
factors participating in ethylene signaling response were identied: EIN3 and EIL1 [67,68]. The physical interaction between FIT
and EIN3/EIL1 is thought to stabilize FIT and trigger up-regulation
of root Fe transport system genes IRT1 and FRO2. Accordingly, a
transcriptome of the ein3 eil1 double mutant under Fe deciency
showed that these genes are involved in the ne tuning of the Fe
deciency response, meeting the photosynthetic needs of the plant
under Fe deciency [69,70]. In A. thaliana, jasmonate was found to
have an inhibitory effect on IRT1 and FRO2 up regulation under Fe
deciency, which is alleviated in the ethylene insensitive mutant
jar1. The repression of IRT1 and FRO2 expression by jasmonate is
independent of FIT suggesting that methyl jasmonate may directly
modulate Fe uptake [71]. In rice, the receptor like protein OsRMC
regulates both jasmonic acid and Fe deciency responses suggesting that in this species jasmonate is positively correlated with Fe
uptake [23].
Interestingly, salicylic acid is both recognized as a defence hormone and a potent Fe chelator [72]. Salicylic acid is even used
for Fe quantication in colorimetric assays [73]. Salicylic acid upregulates genes encoding two Fe responsive transcription factors
bHLH38 and bHLH39 [74]. However, A. thaliana mutants affected in
salicylic acid biosynthesis or salicylic acid signalling do not display
any signicant defect in Fe homeostasis [75]. Nevertheless, several pieces of evidence point to a potential role of salicylic acid in
Fe homeostasis, but the right experimental conditions to highlight
this role still need be to be determined. Using salicylic acid decient
mutants might not be the best way to demonstrate that it acts as

an Fe chelator interfering with plant Fe homeostasis, as the effect

might be masked by potential redundancies with other Fe chelating
systems, such as nicotianamine or citrate. Salicylic acid treatment
of Fe decient peanut plants alleviated Fe deciency and induced
chlorosis [76]. This was associated with an increase in leaf soluble
Fe content. Thus, a possible role of salicylic acid in Fe homeostasis
could rely on its ability to enhance Fe availability, possibly by solubilizing cell wall stocks. Salicylic acid was also found to regulate
the expression of Fe transport genes: A. thaliana YSL3 gene is upregulated by salicylic acid and contributes to defence against the
bacterial pathogen P. syringae pv. tomato DC3000 [77]. More investigations are required to unravel the relationships between salicylic
acid and Fe in modulating immunity.
In addition to these hormones, nitric oxide (NO) modulates
immune responses in very complex ways depending on the radical species that it contributes to form [78,79]. NO is involved in Fe
mobilization in maize and tomato as treatment of plants with NO
donors resulted in a better growth under Fe decient conditions
[80,81]. Tomato plant treatment with NO mediated up-regulation
of the Fe deciency markers LeIRT1 and LeFRO2 via the tomato FIT
homologue FER [81]. This is consistent with the fact that NO Snitrosylates FIT in A. thaliana [82]. However, NO also up-regulates
the expression of AtFER1 gene encoding ferritin, an Fe storage protein dedicated to buffer excess Fe for cell protection [46,83]. One
possible explanation for this apparent contradiction is that Fe deciency leads to mobilization of Fe from internal stores and thus
contributes to locally enhance Fe bioavailability, which may in
turn require ferritin accumulation to prevent oxidative damage.
Alternatively, NO production could occur in different cellular compartments or tissue and affect distinct targets. The role of NO in Fe
homeostasis is clearly important, but the role of NO in the cross
talk between immune responses and Fe homeostasis remains to be
elucidated.
The fact that defense hormone signalling contributes to Fe
homeostasis indicates that ne tuning of Fe trafc, storage and
uptake is important for immunity. During infection, up-regulating
ferritin may be needed to protect the plant cells from oxidative damage caused by Fe released from dying cells. Alternatively,
increased Fe uptake or transport may be required either for fuelling
cells with Fe to reach metabolic needs that could be increased by
pathogen attack, or to enhance ROS accumulation at the site of
infection.

2.5. Microbial siderophores manipulate plant iron homeostasis

and immunity
Siderophores are required for full virulence in many plantpathogens interactions, (for review [84]). In addition to contributing to virulence, some siderophores are able to trigger immune
responses in plants. However, the underlying mechanisms are
still poorly understood. Siderophores may act on immunity in

A. Aznar et al. / Plant Science 240 (2015) 9097

95

Fig. 2. Strategy I and Strategy II: the two different models of Angiosperm iron acquisition mechanisms.
Pathogens trigger pattern triggered immunity via recognition of microbe associated molecular patterns (MAMP) which leads to phenolic accumulation and reactive oxygen
species (ROS) formation. Pathogens secrete siderophores in their hosts and trigger an iron deciency like signal. This signal leads to phenolic accumulation and iron
mobilization and uptake from the rhizosphere. Iron mobilization can be detrimental because Fe catalyses oxidative stress. The plant must deal with this oxidative stress
which can be useful to combat the pathogen but toxic for its proper cells. Ferritin and phenolics have antioxidant effects and contribute to defense against pathogens. Dotted
lines indicate hypothetical positive effect; solid lines indicate processes supported by literature. ROS: reactive oxygen species.

two distinct ways: (i) their Fe scavenging effect which results

in direct activation of defense and (ii) their priming effect for
defense responses, increasing their potency following pathogen
attack. Several lines of evidence indicate that siderophores activate the salicylic acid pathway in A. thaliana by scavenging Fe:
leaf treatment with several apo-siderophores with distinct chemical structures up-regulates the salicylic acid pathway while using
the Fe-siderophore complexes is ineffective [45]. In addition, leaf
treatment with the synthetic Fe chelator EDDHA also triggers
the salicylic acid pathway [44]. Similarly, barley leaf treatment
with the siderophore deferrioxamine triggers up-regulation of PR
and Fe homeostasis gene expression [48]. A transcriptomic analysis of leaves treated with siderophores indicated that the most
over-represented function in differentially expressed genes was
immunity [44]. Siderophore treatment thus clearly mimics a biotic
stress in leaves. This study showed that the major process activated
in roots is heavy metal homeostasis. In addition, siderophores modify Fe distribution at the cellular level [44]. The model proposed
for the mechanism of immunity activation by siderophores via Fe
scavenging involves two steps (Fig. 2). A rst step consists in the
activation of an Fe deciency like response, which in turn results
in a second step consisting in disturbing heavy metal homeostasis.
Fe may not be the only metal involved as its homeostasis is strongly
linked to that of other heavy metals, including copper, zinc and
manganese.
Defense priming by siderophores was described in two monocot
species. Pretreatment with the siderophore coprogen primes maize
defenses against the hemibiotroph pathogenic fungus C. graminicola [85]. Pre-treated maize leaves respond with enhanced PR gene
induction and ROS accumulation upon subsequent attack of the
pathogen [85]. Similar results were obtained with apo-coprogen or

with Fe-coprogen, indicating that priming, in this case, is independent of Fe scavenging. Production of coprogen is required for full
virulence of C. graminicola. The fungus tightly controls coprogen
biosynthesis by repressing it during the early biotrophic phase [85].
Repression of siderophore production during the biotrophic phase
probably allows the pathogen to evade the plants immune system.
Siderophores are thought to be involved in induced systemic resistance by non-pathogenic rhizobacteria [86]. Pretreatment of rice
roots with pseudobactin (Psb374), a siderophore produced by the
systemic resistance inducing strain of P. uorescens WCS374r, provides protection against infection by the rice blast agent M. oryzae
[87]. Furthermore, accelerated defense responses were observed.
Thus, a priming effect by siderophores was observed in both maize
and rice. Whether both priming and eliciting effects can be triggered in the same species by siderophores, and whether this would
provide additive or synergistically enhanced protection remains
to be elucidated. Whether a similar signaling pathway is involved
in immunity activation by siderophores via roots or via aerial
parts, is also an open question. As the priming effect of coprogen
on immune responses in maize does not rely on Fe scavenging,
coprogen may be recognized by a receptor protein activating downstream responses, as in the case for MAMPs that are recognized
by pattern recognition receptors. Animal siderocalin proteins are
able to bind siderophores, thereby blocking their uptake by bacteria
[88]. Whether a similar mechanism exists in plants is still unknown.
Since Graminaceae take up Fe from the rhizosphere via transport
of Fe-phytosiderophore complexes through proteins from the YSL
family [25], perception of microbial siderophores by these plants
may also involve YSL proteins.

96

A. Aznar et al. / Plant Science 240 (2015) 9097

3. Conclusions and open questions

Several lines of evidence highlight the crucial role of plant
Fe homeostasis in plant pathogen-interactions and in particular defense responses. Similar conclusions have been reached in
animals [89], indicating that Fe was conserved as a factor that
potentially modulates defense against invaders in both animal and
plant kingdoms during evolution. Modulating Fe supply leads to
different outcomes depending on the pathogen infection strategy. In some cases, Fe directly contributes to the amplication
of ROS production. Moreover, Fe supply may have an indirect
effect on the metabolic activity of the plant, thereby allowing
production of antimicrobial compounds or other defenses that
require Fe-dependent enzymes. However, Fe deciency also causes
accumulation of antimicrobial compounds. Thus Fe nutrition possibly has opposite effects on immunity depending on specic
plant-pathogen interactions. A comprehensive study comparing
biotroph, hemibiotroph and necrotroph pathogens would allow
a better understanding of the relationship between the pathogen
lifestyle and the role of plant Fe status in the outcome of the infection. In addition, it would be interesting to know whether the plant
Fe status affects pathogen lifestyle or virulence functions, which
could also affect disease progression.
Microbial siderophores initially secreted by pathogens to
acquire Fe from their environment have been shown to trigger
defense responses. Depending on the host, the mechanism of
defense activation by siderophores involves either their Fe scavenging property or receptor mediated recognition as in the case of
pattern triggered immunity. These two mechanisms are not mutually exclusive and have been largely discussed in another review
[90]. More investigation is needed in other models in order to be
able to draw a clear picture of these mechanisms. Furthermore, the
mechanisms used by plants to perceive local Fe depletion induced
by siderophores and translate it into a defense response remains to
be elucidated.
Iron could have a crucial role in the mechanisms of pathogen
perception and downstream signaling defense. It is very likely that
pattern triggered immunity or HR processes recruit Fe transport
proteins that amplify defense signaling via ROS formation. Thus,
it would be of great interest to determine the capacity of various
Fe homeostasis mutants to activate basal defenses and to establish a HR in response to avirulent pathogens. Moreover, secreted
pathogen effectors generally target host functions to favor infection. Thus, it is possible that plant Fe homeostasis genes/proteins
could be targeted by some pathogen effectors. In this context,
siderophores secreted by invaders could participate to the ne
tuning of the infection process in conjunction with effectors. The
only data we have about siderophores show that these molecules
may contribute to virulence and may be detected by the plants
as elicitors. However, it would be interesting to study the role of
siderophores in the modulation of pathogen effectors actions on
the plant. This could be performed by using effector/siderophore
double mutants or by co-injecting effectors and siderophores in
plants.
Altogether, this review argues that Fe should be considered as a
crucial element with complex roles in plant-pathogen interactions.
Important impacts of these ndings could be transferred to crops
whereby Fe homeostasis genes could be underlying disease resistance QTLs. Crops could be engineered to express Fe homeostasis
genes that have strong effect on tolerance to pathogens without affecting yields. Furthermore, plants treated with siderophores
exhibit a transient Fe deciency and immune responses in the
absence of any Fe deciency symptoms. Therefore, siderophores
could be used as natural plant defense stimulators for sustainable
crop protection strategies. To this aim, it will be important to test

whether siderophore treatment limits crop yields, as well as to

evaluate the cost efciency of this strategy.

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