Download Nutrition acquisition strategies during fungal infection of plants

MINIREVIEW
Nutrition acquisition strategies during fungal infection of plants
Hege H. Divon1,2 & Robert Fluhr3
1
Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway; 2Norwegian Institute for Agricultural and
Environmental Research - Bioforsk, Plant Health and Plant Protection Division, Ås, Norway; and 3Department of Plant Science, Weizmann Institute of
Science, Rehovot, Israel
Correspondence: Hege H. Divon,
Department of Plant and Environmental
Sciences, Norwegian University of Life
Sciences, PO Box 5003, N-1432 Ås, Norway.
Tel.: 147 6496 5559; fax: 147 6496 5901;
e-mail: [email protected]
Received 25 August 2006; revised 8 October
2006; accepted 9 October 2006.
DOI:10.1111/j.1574-6968.2006.00504.x
Editor: Richard Staples
Keywords
plant pathogen; fungal nutrition; nitrogen and
carbon regulation.
Abstract
In host–pathogen interactions, efficient pathogen nutrition is a prerequisite for
successful colonization and fungal fitness. Filamentous fungi have a remarkable
capability to adapt and exploit the external nutrient environment. For phytopathogenic fungi, this asset has developed within the context of host physiology
and metabolism. The understanding of nutrient acquisition and pathogen primary
metabolism is of great importance in the development of novel disease control
strategies. In this review, we discuss the current knowledge on how plant nutrient
supplies are utilized by phytopathogenic fungi, and how these activities are
controlled. The generation and use of auxotrophic mutants have been elemental
to the determination of essential and nonessential nutrient compounds from the
plant. Considerable evidence indicates that pathogen entrainment of host metabolism is a widespread phenomenon and can be accomplished by rerouting of the
plant’s responses. Crucial fungal signalling components for nutrient-sensing pathways as well as their developmental dependency have now been identified, and
were shown to operate in a coordinate cross-talk fashion that ensures proper
nutrition-related behaviour during the infection process.
Introduction
Parasitism defines the state in which one organism benefits
at the expense of another. An inherent part of the success of
the parasite in this misbalanced relationship is the securing
of its nourishment. Much research has focused towards
understanding pathogenicity and virulence factors. These
include study of avirulence factors, toxins, cell wall-degrading enzymes (CWDE) and other mechanisms directly linked
to the ability of the pathogen to obtain access to its host. A
less-studied aspect of this interaction is the appreciation of
how nutrients are acquired and how the parasite adapts to
changing nutritional environments. Ultimately, a pathogenic lifestyle is characterized by metabolic dependency on
the host. This is a fine-tuned association where host
physiology is exploited and modified to optimize production of nutrients for the pathogen, and reciprocally pathogen metabolism is adapted to utilize what is made available.
The final fitness of the fungus in this metabolic ‘tug-of-war’
will determine the success of colonization.
In this review, we examine in planta nutrition by plant–pathogenic fungi as established by biochemical and moleFEMS Microbiol Lett XX (2007) 000–000
cular tools. Many of the general concepts have been adopted
from studies in saprophytes such as Saccharomyces cerevisiae, Aspergillus nidulans and Neurospora crassa. The central
questions are as follows: what nutrients are acquired by the
pathogen from the host, what metabolic adjustments have to
be made during the pathogen’s life cycle and what are the
regulatory mechanisms that facilitate this adaptation?
Dynamic nutrient requirements
throughout the infection cycle
Fungal plant pathogens have been traditionally classified by
their lifestyle into biotrophic and necrotrophic types of
parasitism. Biotrophic pathogens are defined by a total
dependency on the host to complete their life cycle, deriving
nutrients from living host cells by differentiation of specialized infection structures called haustoria (Mendgen &
Hahn, 2002). In contrast, necrotrophs do not possess
specialized infection structures and derive nutrients from
sacrificed cells (Lewis, 1973). To accommodate further
diversification of lifestyles, the term ‘hemibiotrophic’ has
been coined to consider situations in which the pathogen
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2
Fig. 1. Infectious phases in phytopathogenic fungi that are relevant to
nutrition acquisition strategies during fungal infection of plants. The
penetration and sporulation phases are common to all fungi. Hemibiotrophs are distinguished by going through successive biotrophic and
necrotrophic phases. Necrotrophs do not have a biotrophic phase,
whereas obligate biotrophs complete their life cycle in this phase,
without proceeding to necrotrophic stage.
renders its host largely alive while establishing itself within
the host tissue. Only after this brief biotrophic-like phase
does it switch to a necrotrophic lifestyle (Perfect & Green,
2001). The infection process itself can be separated into
germination, proliferation and sporulation (Solomon et al.,
2003). The utility of this division is used here by following
the dynamic nutritional challenges facing the pathogen in
the context of its respective infectious phases (Fig. 1).
Spore germination and penetration
Pathogen penetration of the host is facilitated by direct local
mechanical pressure during appressorium development,
enzymatic digestion of plant barriers or by the more
serendipitous availability of stomata, wounds and cracks at
the plant environment interface. It is generally assumed that
during spore germination and the penetration phase, the
phytopathogenic fungi are in a state of starvation and are
completely reliant on nutrients derived from internal stores.
The major components of fungal spore stores include
glycogen, trehalose, polyols such as mannitol and lipids
(Thevelein, 1984; Thines et al., 2000; Voegele et al., 2005).
Recent evidence from several plant–pathogen systems has
focused on the role of fatty acid mobilization during the
penetration process (Table 1). Mobilization of lipid stores
occurs through lipolysis and b-oxidation cycles to form
acetyl-CoA, which is further assimilated into the tricarboxylic acid cycle (TCA cycle) via the glyoxylate cycle. The
glyoxisomal enzyme isocitrate lyase (ICL), a marker for
lipolytic activity, is induced in the absence of glucose and
during growth on gluconeogenic carbon sources such as
acetate and ethanol, and on fatty acids (Bowyer et al., 2000).
During infection, ICL promoter activity was detected predominantly in the prepenetration stage on the plant surface
and declined after appressorial penetration, indicating a
switch from gluconeogenic to glycolytic metabolism at this
stage. Several mutants in which virulence is reduced were
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H.H. Divon & R. Fluhr
found to be defective in peroxisomal development (Kimura
et al., 2001), or disrupted in the glyoxylate cycle enzymes
ICL (Idnurm & Howlett, 2002; Wang et al., 2003) and
malate synthase (MLS; Solomon et al., 2004a). The findings
indicate a universal role for fatty acid metabolism and
glyoxylate cycle during plant penetration, regardless of
pathogenic lifestyle. Interestingly, in some studies, glucose
was able to complement the loss of pathogenesis (Kimura
et al., 2001; Idnurm & Howlett, 2002; Solomon et al.,
2004a). The implications are that a critical role for the fatty
acid metabolic activity is the support of the TCA cycle
through acetyl-CoA production in order to generate ATP
under the poor nutrient conditions that exist at the plant
surface. An additional role for fatty acid metabolism in
glycerol production was demonstrated in the ICL mutant in
Magnaporthe grisea, which shows delayed appressorium
turgor generation (Wang et al., 2003).
Nitrogen constitutes a smaller part of the storage compounds in the spore and might represent a rate-limiting factor
during penetration. Nevertheless, elevated expression of a di/
tri-peptide transporter (Ptr2) at the prepenetration stage in
Stagonospora nodorum is not essential for pathogenicity
(Solomon et al., 2004b), suggesting that recycling of internal
stores can serve as the primary source of nitrogen essentials at
this stage. Indeed, a recent transcriptome survey of barley
powdery mildew revealed a large number of genes involved in
amino acid metabolism, protein turnover and amino acid
recycling, which were expressed during spore germination
and appressorium formation (Thomas et al., 2001).
Biotrophic growth phase and the entrainment
of plant metabolism
Once inside the host, and with internal stores likely exhausted, the pathogen needs to establish itself rapidly by
mobilizing mechanisms that will ensure adequate nutrient
uptake from the host. For the obligate biotrophs, this phase
is initiated by the formation of a haustorium (Voegele, 2006,
and references therein). For hemibiotrophs, deficient in
such feeding structures, some recent evidence suggests that
they have developed mechanisms that entrain host metabolism to establish new sink tissues to suit the needs of the
pathogen. Invertases and hexose sugar transporters play a
key role in defining a leaf as a ‘source’ or as a ‘sink’ of
carbohydrates. The up-regulation of plant extracellular invertase appears to be a common response to various biotic
and abiotic stress-related stimuli, serving to fuel plant
defence responses in the infected tissue (reviewed in Biemelt
& Sonnewald, 2006). This defence strategy is used by
Arabidopsis; however, during powdery mildew infection,
this physiological response of the plant is exploited by the
fungus to ensure a ready supply of carbohydrates for its own
use (Fig. 2; Fotopoulos et al., 2003).
FEMS Microbiol Lett XX (2007) 000–000
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Nutrition acquisition strategies during fungal infection
Table 1. Fungal nutrition-related genes and ESTs expressed during plant infection. Signalling components are not included
Gene/EST name
Acc. No.
Fungal species
Similarity
Function
EP
References
Biotrophic fungi
Aat1 (Pig27)
Aat2 (Pig2)
Ard1
Bgl1
Gat1
Gltrn1
GS-1
Hxt1
Met6
Mad1 (Pig8)
Pma
AJ308252
U81794
AJ809335
AJ575269
AF271266
AF376000
AF395112
AJ310209
AF226997
O00058
AF406807
U. fabae
U. fabae
U. fabae
U. fabae
C. fulvum
B. graminis
B. graminis
U. fabae
C. fulvum
U. fabae
B. graminis
Histidine uptake
Amino acid transport
D-arabitol biosynthesis
Cellobiose degradation
GABA metabolism
Glucose uptake
Glutamine synthesis
Uptake of D-glc, D-frc
Methionine biosynthesis
Mannitol metabolism
Nutrient uptake
ND
ND
ND
ND
ND
ND
ND
ND
Yes
ND
ND
Struck et al. (2002)
Hahn et al. (1997)
Link et al. (2005)
Haerter & Voegele (2004)
Solomon & Oliver (2002)
Zhang et al. (2005)
Zhang et al. (2005)
Voegele et al. (2001)
Solomon et al. (2000)
Voegele et al. (2005)
Zhang et al. (2005)
pSI-9
pSI-10
Thi1 (Pig1)
Y14555
Y14556
AJ250426
C. fulvum
C. fulvum
U. fabae
Carbon metabolism
Carbon metabolism
Vitamin B1 biosynthesis
ND
ND
ND
Thi2 (Pig4)
AJ250427
U. fabae
Amino acid transporter
Amino acid transporter
D-arabitol dehydrogenase 1
b-glucosidase
GABA transaminase
Glucose transporter
Glutamine synthetase
Hexose transporter
Methionine synthase
Mannitol dehydrogenase
Plasma membrane
H1 ATPase
Aldehyde dehydrogenase
Alcohol dehydrogenase
Pyrimidine biosynthetic
enzyme
Thiazole biosynthetic enzyme
Vitamin B1 biosynthesis
ND
Tr4
BU672643
P. triticina
Vitamin B1 biosynthesis
ND
Tr21
UfAat3
UfInv1
Uf-Pma1
BU672660
AJ628939
AJ640083
AJ003067
P. triticina
U. fabae
U. fabae
U. fabae
Pyrimidine biosynthetic
enzyme
Thiazole biosynthetic enzyme
Amino acid transporter
Invertase
Plasma membrane
H1 ATPase
Coleman et al. (1997)
Coleman et al. (1997)
Sohn et al. (2000),
Hahn & Mendgen (1997)
Sohn et al. (2000),
Hahn & Mendgen (1997)
Thara et al. (2003)
Vitamin B1 biosynthesis
leu, met, cys uptake
Sugar hydrolysis
Proton-driven nutrient
uptake
ND
ND
ND
ND
Thara et al. (2003)
Struck et al. (2004)
Voegele et al. (2006)
Struck et al. (1996),
Struck et al. (1998)
Hemi-biotrophic fungi
Arg1
AB045736
Gap1 (Fol5B10)
CK615491
Gpdh
AY331190
Icl1
Mtd1 (Fol8D6)
pCgGS
Uricase (Fol6D12)
Necrotrophic fungi
Als1
AY118108
CK615494
L78067
CK615495
F. oxysporum
Argininosuccinate lyase
Arginine biosynthesis
F. oxysporum
General amino acid permease Amino acid uptake
C. gloeosporioides Glycerol-3-phosphate
Carbon utilization
dehydrogenase
L. maculans
Isocitrate lyase
Glyoxylate cycle
F. oxysporum
Oligopeptide transporter
Peptide uptake
C. gloeosporioides Glutamine synthetase
Glutamine synthesis
F. oxysporum
Uricase
Uric acid anabolism
DQ167577
S. nodorum
Cbl1
Glo1
Icl1
Mls1
Mpd1
EAA68828
AY576607
AF540383
AY508881
AY587541
F. graminearum
S. nodorum
M. grisea
S. nodorum
S. nodorum
Msy1
Odc1
Pth2
Pth3
EAA75179
AJ249387
AF027979
O42621
F. graminearum
S. nodorum
M. grisea
M. grisea
Ptr2
AY187281
BM135467
S. nodorum
F. graminearum
BM137184
MG03533
MG04385
MG05526
F. graminearum
M. grisea
M. grisea
M. grisea
Delta-aminolaevulinic acid
synthase
Cystathionine betalyase
Glyoxylase I
Isocitrate lyase
Malate synthase
Mannitol 1-phosphate
dehydrogenase
Methionine synthase
Ornithine decarboxylase
Carnitine acetyltranferase
Imidazoleglycerol-P
dehydratase
di/tripeptide transporter
NADP-specific glutamate
dehydrogenase
Hexose transporter protein
Formamidase
Urea transporter
Methylammonium permease
Yes
ND
No
Namiki et al. (2001)
Divon et al. (2005)
Wei et al. (2004)
Yes
ND
ND
ND
Idnurm & Howlett, 2002
Divon et al. (2005)
Stephenson et al. (1997)
Divon et al. (2005)
Heme biosynthesis
Yes
Solomon et al. (2006)
Methionine biosynthesis
Glycolytic bypass pathway
Glyoxylate cycle
Glyoxylate cycle
Mannitol metabolism
Yes
No
Yes
Yes
Yes
Seong et al. (2005)
Solomon & Oliver (2004)
Wang et al. (2003)
Solomon et al. (2004a)
Solomon et al. (2005)
Methionine biosynthesis
Polyamine biosynthesis
Fatty acid transport
Histidine biosynthesis
Yes
Yes
Yes
Yes
Seong et al. (2005)
Bailey et al. (2000)
Sweigard et al. (1998)
Sweigard et al. (1998)
di/tri peptide uptake
Glutamate synthesis
No
ND
Solomon et al. (2004b)
Kruger et al. (2002)
Hexose uptake
trp catabolism
Urea uptake
Nitrogen sensing and
uptake
ND
ND
ND
ND
Kruger et al. (2002)
Donofrio et al. (2006)
Donofrio et al. (2006)
Donofrio et al. (2006)
EP, effects of mutation on pathogenicity.
The reader is referred to Guldener et al. (2006) and Jakupovic et al. (2006), for additional genes expressed in planta in F. graminearum and U. fabae,
respectively.
FEMS Microbiol Lett XX (2007) 000–000
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H.H. Divon & R. Fluhr
Fig. 2. Examples of the entrainment of plant metabolism by phytopathogenic fungi. Sensing of nitrogen or carbon availability (NS, CS; flash arrow), or
the absence of preferable nutrient sources, triggers signal transduction pathways that initiate transcription of transporters and pathway-specific
metabolic enzymes. The de-repression of these pathways is regulated by de-phosphorylation and binding of AREA to nitrogen catabolite-regulated
gene promoters (Ravagnani et al., 1997; Beck & Hall, 1999), and by SNF1-dependent phosphorylation and release of CREA repressor from carbon
catabolite-regulated gene promoters (DeVit et al., 1997; Gancedo, 1998). Plant resistance responses (red arrows), involving ornithine or ROS scavengers
such as GABA, are taken up through amino acid (AA) permeases and metabolized by the fungus (Oliver & Solomon, 2004; Guldener et al., 2006). Uric
acid, the product of disease-induced enhanced catabolic processes in the plant, can serve as a fungal nutrient by conversion to allantoin by uricase and
subsequent recovery of ammonia (Divon et al., 2005). Plant invertases that are induced by the plant to fuel its cellular defence needs, as well as CWDE
secreted by the pathogen, will produce depolymerized sugars that the fungus can utilize (Ospina-Giraldo et al., 2003; Biemelt & Sonnewald, 2006). PM,
plasma membrane; CW, cell wall.
In a manner similar to the utilization of plant carbohydrates during infection, nitrogen acquisition can also be
subverted for fungal benefit as illustrated by the metabolism
of g-amino butyric acid (GABA) during Cladosporium
fulvum infection of tomato (Solomon & Oliver, 2001).
GABA, normally a major amino-acid constituent of the
tomato apoplast, accumulates to even higher (millimolar)
levels during the compatible interaction. The accumulation
was concomitant with the induction of tomato glutamate
decarboxylase (GAD) responsible for GABA synthesis, and
the fungal GABA transaminase (Gat1; Table 1), metabolizing
GABA into succinic semialdehyde (Solomon & Oliver,
2002). The utilization of GABA as a nutrient has so far only
been demonstrated for Cladosporium fulvum, and may be
part of the adaptation to a biotrophic lifestyle without
haustoria, which occurs exclusively in the apoplastic spaces
of the leaf. Interestingly, the ramifications of modulation of
GABA metabolism may be more subtle. The GABA shunt
can also serve as part of a cellular scavenger system for
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oxidative stress signals. The role of reactive oxygen species
(ROS) in plant defence signalling is well established, and one
can speculate that as in the case of carbohydrate acquisition
successful colonization will subvert plant signals for defence
to provide a source of nutrients for pathogenic fungi (Fig. 2;
Oliver & Solomon, 2004). A similar scenario may occur in
the Fusarium oxysporum interaction with tomato, where
uricase, an enzyme catalysing the conversion of uric
acid to allantoin, was induced during the early nonnecrotic
stages of infection (Table 1; Divon et al., 2005). Uric acid can
act as a scavenger of ROS (Becker et al., 1989) and like
GABA may serve the plant in that role. Hence, the fungus
may utilize uric acid derived from host catabolism and
defence reactions (Fig. 2). Additional evidence for the
role of metabolites with scavenging capacity comes
from a study of the rust fungus Uromyces fabae. Voegele
et al. (2005) suggested that the pathogen-derived compound
mannitol, which the host, Vicia faba, can neither produce
nor metabolize, accumulates in infected leaves to serve as a
FEMS Microbiol Lett XX (2007) 000–000
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Nutrition acquisition strategies during fungal infection
ROS scavenger to protect the pathogen in the infection
process.
While great strides have been made in understanding how
bacterial pathogen effectors are delivered through the type
III secretion systems found in plant–pathogenic bacteria
interactions, less is known at the molecular level of how
fungi entrain host metabolism. For example, in the Cladosporium fulvum interaction with tomato, an extracellular
fungal protein was detected that modifies a plant cysteine
protease and subsequent plant defence responses (Rooney
et al., 2005). Interestingly, proteins from the rust fungi
Melamspora lini and U. fabae are localized to the
host cytoplasm (Dodds et al., 2004; Kemen et al., 2005).
This may indicate that fungal effector proteins can gain
intracellular access and thus target cellular metabolic
crossroads.
Necrotrophic growth phase and entrainment of
plant metabolism
Shortly after penetration, necrotrophic plant pathogenic
fungi, aided by production of toxins, hydrolytic enzymes
and necrosis-related proteins, cause cell lysis and death of
host cells. One may surmise that the lysis of host cells would
lead to an increase in accessible nutrients; however, as
evidenced from examination of auxotrophic mutants, certain amino acids such as methionine and histidine are not
supplied in sufficient quantity by the host tissue and their
biosynthesis is carried out by the fungus (Sweigard et al.,
1998; Balhadere et al., 1999; Seong et al., 2005). The
coordinate expression of nutrient acquisition genes during
infection that includes possible sources for amino acid
backbones was revealed in the transcriptome analysis of
Fusarium graminearum infection (Guldener et al., 2006).
In this case, transporters of sugars and nitrogenous
compounds were expressed in early infection, including
an ornithine transporter expressed from day 2 postinfection.
Ornithine is a nonprotein amino acid and an important
intermediate in metabolic pathways such as arginine and
proline biosynthesis, as well as the biosynthesis of putrescine, a precursor for other polyamines and pyrimidines.
Ornithine uptake was followed by expression of arginine,
proline and putrescine biosynthetic genes from day 3 to 6
postinfection (arginine: ornithine carbamoyltransferase,
carbamoyl-P synthase and acetylglutamate kinase; proline:
ornithine aminotransferase; putrescine: ornithine decarboxylase). In line with this observation, a putrescine auxotrophic mutant from the wheat pathogen Stagonospora
nodorum, which was disrupted in the gene encoding ornithine decarboxylase, odc1, was found to have greatly
reduced virulence on wheat (Table 1; Bailey et al., 2000).
Ornithine, as a precursor in polyamine synthesis, is involved
in plant stress responses (Walters, 2003). Hence, we enFEMS Microbiol Lett XX (2007) 000–000
counter, as in the case of carbohydrates, GABA and uric acid
metabolism, the concept that successful pathogens hijack
potential plant defences for their nutritional benefit during
colonization (Fig. 2).
Sporulation
Sporulation marks the completion of the pathogenic life
cycle. True biotrophic fungi complete their life cycle by
maintaining a biotrophic state, whereas hemibiotrophs and
necrotrophs complete their life cycle in a necrotrophic state
(Fig. 1). In vitro studies have shown that sporulation can be
triggered by exhaustion of nitrogen and carbon sources
(Griffin, 1994). Thus, in a manner analogous to the finding
that fungi undergo nutrient limitation during the initial
penetration, it may be anticipated that nutrient limitation
triggers sporulation. However, experiments describing the in
planta sporulation, from the hemibiotrophic interaction of
Mycosphaerella graminicola with wheat, points to a different
situation. cDNA microarray analysis during in planta
growth revealed fungal expression of a subset of genes that
resemble transcripts associated with in vitro growth in rich
medium rather than poor medium (Keon et al., 2005).
The results suggest an alternative trigger for the sporulation event such as, for example, a quorum-sensing-like
mechanism.
A particularly illuminating example of dependence on
nutrient transport in the late growth stages was revealed in
the glycerol-3-phosphate dehydrogenase (Gpdh) disruption
mutant of the hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae (Table 1; Wei et al., 2004).
The mutant contains reduced amounts of glycerol, and
during heterotrophic growth on defined media, the mutant
strain exhibited severe defects in carbon utilization and
failed to conidiate. This defect could be complemented by
exogenous addition of glycerol. Surprisingly, in spite of these
profound growth defects during heterotrophic growth, the
gpdh mutant strain displayed normal pathogenicity in
planta, progressing normally through infection and conidiation. Importantly, the analysis of plant tissues showed
significant depletion of glycerol in the infection zone,
indicating glycerol transfer from the plant to the fungal
pathogen.
Regulation of fungal nutrition gene
expression during infection
Nutrient availability in the external environment dictates
expression of fungal nutrition genes via general metabolic
processes known as catabolite repression. This process
implies that, as long as sufficient amounts of a favourable
nutrient source are available, transcription of alternative
uptake systems and catabolic enzymes is repressed. The
preferred source for the carbon-regulatory system is glucose,
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6
and it is ammonia and glutamine for the nitrogen-regulatory system. Catabolite repression has been extensively
studied in saprobes like Saccharomyces cerevisiae, N. crassa
and A. nidulans (Marzluf, 1997; Carlson, 1998; Gancedo,
1998; Magasanik & Kaiser, 2002), and given the high degree
of conservation in this process, these saprobes have served as
models to guide similar research in phytopathogenic fungi.
Carbon catabolite repression (CCR)
CCR signifies the transcriptional regulation of genes involved in utilization of alternative carbon sources. It is
driven by glucose repression and substrate induction. In
Saccharomyces cerevisiae, the SNF1 (sucrose nonfermenting
1) gene is required for expression of catabolite-repressed
genes (Fig. 2; Gancedo, 1998, and references therein). It
encodes a protein kinase whose major function is to
phosphorylate Mig1, a DNA-binding transcriptional repressor. When glucose is limiting, phosphorylation of Mig1
causes its dissociation from the promoters of repressed
genes (DeVit et al., 1997), thus motivating the release from
CCR. SNF1-orthologues have been cloned from the maize
pathogen Cochliobolus carbonum (CcSnf1; Tonukari et al.,
2000) and the vascular wilt pathogen F. oxysporum (FoSnf1;
Ospina-Giraldo et al., 2003). Disruption mutants were
compromised for growth on secondary carbon sources, and
production of several secreted CWDE was reduced with a
concomitant reduction in virulence. This phenomenon was
observed at the penetration stage (Tonukari et al., 2000), as
well as in later stages of plant colonization (Ospina-Giraldo
et al., 2003). Thus, CCR is directly linked to pathogenicity.
In addition, SNF1 might facilitate de-repression of hexose
transporters for uptake and utilization of sugars released
from degraded cell wall polymers (Fig. 2; Carlson, 1998).
The orthologue of MIG1 in filamentous fungi is called CreA,
encoding a repressor with two zinc fingers of the C2H2 class
(Fig. 2; Ronne, 1995). CreA genes were isolated from the
plant pathogenic fungi Gibberella fujikuroi and Botrytis
cinerea (Tudzynski et al., 2000); however, their contribution
to nutrient acquisition during infection was not determined.
In another approach, random insertional mutagenesis in the
hemibiotroph Colletotrichum lindemuthianum identified a
mutant disrupted in the metabolic switch between the
biotrophic and the necrotrophic phases (Dufresne et al.,
2000). The corresponding gene, encoding a putative zinc
finger GAL4-like transcriptional activator, was designated
Clta1.
Nitrogen catabolite repression (NCR) and the
‘nitrogen starvation hypothesis’
The hypothesis that plant–pathogenic fungi experience
nitrogen nutrient limitation during in planta growth (Snoeijers et al., 2000) is based on observations that showed the
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H.H. Divon & R. Fluhr
induction of starvation-regulated genes in Cladosporium
fulvum, Magnaporthe grisea and others during the infection
process (Talbot et al., 1993; Van den Ackerveken et al., 1994;
Coleman et al., 1997; Stephenson et al., 1997; Snoeijers et al.,
2000). NCR embodies the transcriptional regulation of
permeases and catabolic enzymes needed for the utilization
of secondary nitrogen sources. In yeast, the gene GLN3
encodes a global activator of the GATA-type zinc finger
family, recognizing the core consensus sequence 5 0 - (A/T/
C)GATA(A/G) -3 0 found in NCR-regulated promoters (Ravagnani et al., 1997; Starich et al., 1998). In the presence of
favourable nitrogen sources such as ammonia and glutamine, Gln3, phosphorylated by the TOR (target of rapamycin) kinase (Beck & Hall, 1999; Fitzgibbon et al., 2005), is
sequestered to the cytosol by inhibitory binding to the
negative regulator Nil2. During nitrogen starvation, due to
the lack of favourable nitrogen sources, de-phosphorylation
of Gln3 relieves it from Nil2 repression, and it relocates to
the nucleus where it activates transcription of NCR-controlled genes, either alone or in combination with pathwayspecific activators (Marzluf, 1997; Magasanik & Kaiser,
2002). Once identified as a key regulator of nitrogen
metabolism, orthologues of GLN3 have been extensively
studied in saprobes like A. nidulans and N. crassa (AreA and
nit-2, respectively; Marzluf, 1997), and in several phytopathogenic fungi. They act as central control devices aiding
the fungus in adapting to different nutrient environments
(Fig. 2).
As the nutrient environment varies with the pathogenic
lifestyle, the dependency on AREA-like regulators during
infection is expected to vary accordingly. Disruption of
Clnr1 in the hemibiotrophic fungus Colletotrichum lindemuthianum severely compromised the infection cycle at the
stage of secondary hyphae formation and almost completely
eliminated the occurrence of anthracnose symptoms (Pellier
et al., 2003). Disruption of Fnr1 in F. oxysporum caused a
delay in disease symptom development, attributed in part to
the abrogated expression of nutrition genes normally upregulated during in planta growth (Divon et al., 2006).
Conversely, disruption of Nut1 in the rice blast fungus,
Magnaporthe grisea, had only a minor effect on virulence
(Froeliger & Carpenter, 1996). In Cladosporium fulvum,
recent evidence shows that deletion of the Nrf1 gene
significantly reduces Cladosporium fulvum virulence on
tomato; however, this reduction is not connected to the
abolished expression of Avr9 (Thomma et al., 2006). Interestingly, GABA uptake via the GABA permease in A.
nidulans is subject to NCR (Hutchings et al., 1999). Under
the assumption that GABA constitutes a significant nutrient
for Cladosporium fulvum during pathogenicity (Solomon &
Oliver, 2002), it is likely that loss of virulence in the nrf1
mutant is, at least in part, caused by nutrient deficiency due
to the inability to take up and metabolize GABA.
FEMS Microbiol Lett XX (2007) 000–000
7
Nutrition acquisition strategies during fungal infection
Based on the existing evidence, it is clear that the main
impact of AREA on pathogenic ability is through the
regulation of nutrition. Interestingly, in hemibiotrophs, the
impact of AREA on regulation seems to somewhat correlate
with the ‘degree’ of biotrophy, i.e. the time spent in the plant
apoplast. Thus, in Magnaporthe grisea, which grows necrotrophically almost immediately after penetration, the effect
of AreA disruption is minor (Froeliger & Carpenter, 1996).
In contrast, in the hemibiotrophs F. oxysporum, Colletotrichum lindemuthianum and Cladosporium Fulvum, such a
disruption shows a clear effect (Pellier et al., 2003; Divon
et al., 2006; Thomma et al., 2006). Apparently, hemibiotrophs that have an extended growth period in the nutrientpoor apoplast will tend to rely on AREA-regulatory nutrient
control. In comparison, this regulatory control will be less
important in the obligate biotrophic pathogens that feed
through specialized infection structures or necrotrophs to
whom ample nutrients are available. Finally, during in
planta growth, nitrogen starvation might be seen as a stagespecific or recurring condition, rather than a continuous
condition. Transition periods from one infectious phase to
the next, i.e. germination, sporulation and transition between the biotrophic and necrotrophic phase, are likely to
increase the demand for energy and metabolite precursors
and require precise regulation.
grisea (Thines et al., 2000). In the MAP kinase mutant,
Dpmk1, appressorium formation is impaired and glycogen
and lipid mobilization do not occur during germination.
The same processes were also retarded markedly in a
cAMP-dependent protein kinase A (PKA) mutant (DcpkA).
Furthermore, in a Dmac1 sum1-99 mutant, carrying a
mutation in the regulatory subunit of PKA that renders it
cAMP-independent and nearly constitutively active, glycogen and lipid degradation were rapid and preceded appressorium morphogenesis (Thines et al., 2000). In another
example, appressorium formation was also impaired in the
nir1 mutant in Colletotrichum acutatum (Horowitz et al.,
2006). Nir1, an orthologue of NirA in A. nidulans, is the
pathway-specific activator of nitrate utilization genes, and
the Nir1 mutation abolished activation of nitrate and nitrite
reductase and was assumed to account partly for the
penetration-defect phenotype. However, the nir1 mutant
displayed additional appressorium developmental defects
not present in nitrate reductase mutants (Horowitz et al.,
2006). Together, these results indicate that considerable
crosstalk occurs during development of the functional
appressorium, involving both traditional catabolite-repressed regulatory pathways, MAP kinase pathways and
cAMP-dependent PKA (Fig. 3).
Linkage of nutritional regulation and
development
Conclusions
Apart from the influence of the external environment, stagespecific requirements for nutrients are also linked to the
development of specialized infection structures. This is
illustrated by the fact that, in obligate biotrophic fungi,
nutrition genes are insensitive to the exogenous nutrient
availability, and instead their transcriptional control is
linked to the development of haustoria. For example,
thiamine biosynthetic genes, thi1 and thi2, in the rust fungus
U. fabae do not respond with down-regulation to exogenously applied thiamine as do saprophytic fungi (Sohn et al.,
2000). Similarly, appressorium turgor generation depends
on accumulation of large quantities of glycerol, which is
acquired from breakdown of internal stores of lipids
and glycogen (Thines et al., 2000). Such processes are
usually under CCR (Hardie et al., 1998); however, impairment of the CCR regulatory pathway in Cochliobolus
carbonum by disruption of Snf1 did not affect appressorium
formation and turgor generation (Tonukari et al., 2000; see
below), indicating that other mechanisms override SNF1
regulation of fatty acid breakdown and peroxisomal
function.
The complexity of metabolic and developmental interaction was investigated in the process of turgor generation in
developmental, nonpathogenic mutants in Magnaporthe
FEMS Microbiol Lett XX (2007) 000–000
An understanding of fungal metabolism and adaptation
during host colonization is of critical importance to evaluate
fully the complex orchestration of the activities that rule
host–pathogen interactions. This knowledge will provide a
rational basis for the development of new disease control
strategies. Studies of gene expression and auxotrophic
mutants have provided considerable information regarding
those nutrients available from the plant and those that are
not. Elaborate mechanisms have been unravelled that indicate how host and pathogen compete for nutrient supplies, and how plant defence mechanisms can be exploited
by fungi for nutritional purposes. Besides amino acids, the
nonprotein amino acids GABA and ornithine stand out as
potential providers of nitrogen, and as precursors in biosynthetic pathways for compounds not adequately provided by
the plant. More case studies will be needed to determine
whether these represent specific or global mechanisms. Key
regulatory factors involved in nutrient signalling and gene
expression have been characterized. Although their impact
on pathogenicity appears to be species specific, commonalties between lifestyle preference and regulatory control are
now emerging. While important advances have been made
in our ability to describe the metabolic events surrounding
fungal ingress, little is known at the molecular level of how
fungi entrain host metabolism. This accentuates the future
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
8
H.H. Divon & R. Fluhr
Fig. 3. Nutrition-related processes that take place during appressorium-mediated penetration. MAP kinase- and cAMP-dependent PKA signalling
pathways are involved in mobilization of glycogen and lipid stores from the spore. Turgor generation is accomplished by compartmentalization and
rapid degradation of lipid and glycogen reserves in the appressorium (Thines et al., 2000). The glyoxylate cycle (Glc) is involved in turgor generation, but
also in replenishing intermediates to the TCA cycle from acetyl-CoA generated in fatty acid oxidation (Kimura et al., 2001; Idnurm & Howlett, 2002;
Wang et al., 2003; Solomon et al., 2004a). An NirA-like transcription factor, Nir1, is required for transcription of nitrate reductase (NR), necessary for
nitrate assimilation in Colletotrichum acutatum, but is also involved in additional processes related to appressorium formation (Horowitz et al., 2006).
The grey/white arrow in the germ tube indicates transport of glycogen and lipid metabolic products; the flash arrow indicates sensing mechanism(s);
and the small white arrows in the appressorium signify turgor generation.
challenge in understanding metabolite requisition by fungal
parasites.
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