Download The zig-zag-zig in oomycete–plant interactions

MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562
DOI: 10.1111/J.1364-3703.2009.00547.X
Review
The zig-zag-zig in oomycete–plant interactions
INGO HEIN 1 , ELEANOR M. GILROY 2 , MILES R. ARMSTRONG 2 AND PAUL R. J. BIRCH 2,3, *
1
Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
3
Division of Plant Science, College of Life Sciences, University of Dundee (at SCRI), Invergowrie, Dundee DD2 5DA, UK
2
SUMMARY
In addition to a range of preformed barriers, plants defend themselves against microbial invasion by detecting conserved,
secreted molecules, called pathogen-associated molecular patterns (PAMPs). PAMP-triggered immunity (PTI) is the first inducible layer of plant defence that microbial pathogens must
navigate by the delivery of effector proteins that act to suppress
or otherwise manipulate key components of resistance. Effectors
may themselves be targeted by a further layer of defence,
effector-triggered immunity (ETI), as their presence inside or
outside host cells may be detected by resistance proteins. This
‘zig-zag-zig’ of tightly co-evolving molecular interactions determines the outcome of attempted infection. In this article, we
consider the complex molecular interplay between plants and
plant pathogenic oomycetes, drawing on recent literature to
illustrate what is known about oomycete PAMPs and elicitors of
defence responses, the effectors they utilize to suppress PTI, and
the phenomenal molecular ‘battle’ between effector and resistance (R) genes that dictates the establishment or evasion of ETI.
INTRODUCTION
Above and below ground, plants face a constant barrage of
invading microorganisms, including bacteria, fungi and
oomycetes. Yet, only a small percentage of plant–pathogen interactions lead to successful disease development, as the majority
of plant species are resistant to invasion by all isolates of any
given microbial species. Pathogen host range is thought to be
limited initially by the ability of a microbe to penetrate preformed barriers, such as the cuticle and cell wall, and to detoxify
constitutively accumulating host antimicrobials (phytoanticipins)
which vary between plant species (Ingle et al., 2006). A pathogen that overcomes these obstructions will encounter the plant
non-self surveillance system that perceives attempted invasions,
*Correspondence: E-mail: [email protected]
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JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD
activating a diverse array of effective, broad-range defences
(Ingle et al., 2006; Zipfel, 2008). Perception initially involves the
detection of conserved molecules (microbe- or pathogenassociated molecular patterns, MAMPs or PAMPs) that are
secreted or displayed on the surface of microorganisms. PAMPtriggered immunity (PTI) constitutes a front-line defence that
must be overcome by microorganisms for successful colonization
of plant tissues (Jones and Dangl, 2006; Zipfel, 2008). This is
achieved by the secretion of virulence determinants, called effectors, which act either outside or inside the host cell to suppress
or otherwise manipulate plant innate immunity. Effectortriggered susceptibility (ETS) includes the suppression of PTI,
representing the first level at which molecular co-evolution
between host and pathogen occurs, as pathogen effectors have
increasingly been shown to suppress immunity via direct
molecular interactions with host defence-associated proteins
(Block et al., 2008; Chisholm et al., 2006; Grant et al., 2006;
Jones and Dangl, 2006).
Plants have a second line of defence, in the form of resistance
(R) proteins, the products of which directly or indirectly detect
effectors (termed avirulence proteins; AVRs), and confer immunity to pathogens that are successful in suppressing PTI. Effectortriggered immunity (ETI) represents a second level at which
host–pathogen molecular co-evolution occurs, as effectors
evolve to evade detection and R proteins evolve to establish or
retain detection (Jones and Dangl, 2006). In this review, we
consider the complex molecular interactions between plants and
plant pathogenic oomycetes, drawing on recent literature to
illustrate what is known about oomycete PAMPs and the effectors they utilize to suppress host immune systems. We also
consider the remarkable molecular evolutionary ‘battle’ between
effectors, their targets in the host and R proteins, which dictate
whether ETI is activated or evaded (Fig. 1).
Plant pathogenic oomycetes
Oomycetes are a diverse group of organisms that morphologically resemble fungi, yet are members of the chromista, more
closely related to key organisms in aquatic environments, such as
brown algae (e.g. kelp), golden-brown algae and diatoms. The
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Fig. 1 The zig-zag-zig in oomycete–plant
interactions (modified from Jones and Dangl,
2006). Shown are the characterized oomycete
pathogen-associated molecular patterns (PAMPs)
and other elicitors of PAMP-triggered immunity
(PTI) and necrosis [represented by a dotted arrow
extending PTI beyond the threshold for host
programmed cell death (PCD)]; examples of
oomycete effectors that contribute to effector
triggered susceptibility (ETS); and examples of
host resistance proteins that detect oomycete
effectors to trigger immunity (ETI). The amplitude
of defence is shown on the y axis, and the
threshold for activation of host PCD is also
indicated. CBEL, cellulose-binding elicitor lectin;
CRN, crinkling and necrosis; NLP, Nep1-like
protein; PRR, pattern recognition receptor; R,
resistance; SCR, small cysteine-rich.
oomycetes include a diverse range of free-living water moulds,
as well as pathogens of plants, algae, insects, fish, crustaceans,
mammals and various microbes, including fungi (Kamoun, 2003,
2006). Plant pathogenic oomycetes cause devastating diseases
of crop, ornamental and native species and are arguably the
most important pathogens of dicotyledenous plants. The most
damaging groups comprise more than 80 species of Phytophthora (Blair et al., 2008), several genera of the downy mildews
(the most important being Peronospora, Plasmopora and
Bremia) (Göker et al., 2006) and more than 110 species of
Pythium (Levesque and de Cock, 2004).
There is great diversity in oomycete infection strategies, from
largely opportunistic or weakly pathogenic necrotrophs with
wide host ranges, typified by many soil-borne Pythium species, to
the highly specialized, biotrophic, aerially disseminated, foliar
downy mildews. The genus Phytophthora spans these two
extremes of lifestyle, including species specialized in colonizing
leaf litter to a range of highly specialized pathogenic hemibiotrophs. A common feature, however, is the absolute dependence on living plant tissue to complete their life cycle.
Oomycetes undergo a series of developmental stages
throughout a successful infection cycle, including the formation
of sporangia, release of motile zoospores, their encystment and
germination to form hyphae and appressoria, the production of
primary and secondary infection hyphae, haustoria and, finally,
sporangiophores (Birch and Cooke, 2004). These various developmental processes facilitate dispersal, recognition of the
host, adhesion, penetration and colonization, encompassing
biotrophic and/or necrotrophic phases of infection, finally
leading once again to dispersal. Three plant pathogenic
oomycetes, in particular, can be considered as models to study
the molecular co-evolution with the host. The soybean pathogen,
Phytophthora sojae, invades and colonizes roots, whereas the
late blight pathogen of potato and tomato is an aerially disseminated, foliar pathogen. Both are hemi-biotrophs. In contrast, the
downy mildew Hyaloperonospora arabidopsidis (formerly Peronospora parasitica) is an obligate biotrophic pathogen of the
model plant Arabidopsis. The genomes of all three have been
sequenced (Lamour et al., 2007; Tyler et al., 2006), which will
help to reveal the full array and diversity of proteinaceous
PAMPs and effectors that trigger and/or suppress the plant
immune system. Examples will be drawn from each to illustrate
the challenges faced by oomycetes in establishing infection, and
in the molecular co-evolution that dictates the outcome of their
interactions with plants.
OOMYCETE PAMPS AND OTHER ELICITORS OF
PLANT IMMUNITY
All classes of microorganism possess characteristic structural
and enzymatic proteins so essential for fitness that they are
indispensable. Some act as PAMPs, recognized as non-self molecules by distinct pattern recognition receptors (PRRs) in plants
in a manner analogous to the Toll- and Toll-like family of receptors of the mammalian innate immune system (Zipfel, 2008). The
consequent PTI comprises a range of universal responses, including ion fluxes, mitogen-activated protein kinase (MAPK) cascades, production of reactive oxygen species (ROS), cell wall
reinforcement and rapid induction of defence genes, largely
regulated by WRKY transcription factors (Ingle et al., 2006). In
order to infect a plant host, successful pathogens must evade
recognition or evolve effectors, some capable of entering the
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Table 1 Selected oomycete secreted proteins that trigger and/or suppress plant immune responses.
Secreted oomycete protein
Acts as a PAMP or
elicitor of necrosis
Elicits ETI
Possible contribution
to virulence
GP42 (transglutaminase family)
Pep-13 PAMP
No
None
CBEL family
Cellulose binding domain PAMP
No
Adhesion to the plant cell
INF1 (member of elicitin family)
Activates PTI and necrosis.
INF1-dependent ROS production
requires SERK3/BAK1
Triggers PTI and necrosis
No
None
No
PcF triggers necrosis and
defence proteins
Triggers necrosis
No
Unknown. Evidence of
diversifying selection
Unknown
Unknown
Facilitates switch from
biotrophy to necrotrophy?
Unknown
No
Unknown
Inhibits host protease P69B
No
Unknown
RXLR effector AVR3a
No
R3a-dependent
RXLR effector Avr1b
No
RPS1b-dependent
RXLR effector ATR1
No
RPP1-dependent
Inhibits host papain proteases
PIP1 and RCR3
Suppresses INF1-mediated
cell death
Suppresses BAX-dependent
cell death
Suppresses PTI
RXLR effector ATR13
No
RPP13-dependent
Suppresses PTI
NLP family
PcF, SCR74 and SCR91
Crinkling and necrosis (Crn) family
Glucanase inhibitors GIP1 and
GIP2
EPI1 (member of Kazal-like
protease inhibitor family)
EPIC2B (member of cystatin family)
Unknown
Inhibits host glucanases
Selected references
Nürnberger et al. (1994);
Brunner et al. (2002)
Gaulin et al. (2006);
Dumas et al. (2008)
Kamoun (2006);
Heese et al. (2007)
Fellbrich et al. (2002);
Qutob et al. (2006).
Orosomando et al. (2001);
Liu et al. (2005)
Torto et al. (2003)
Rose et al. (2002);
Damasceno et al. (2008)
Tian et al. (2004)
Tian et al. (2007);
Song et al. (2009)
Armstrong et al. (2005);
Bos et al. (2006)
Shan et al. (2004); Dou
et al. (2008b)
Rehmany et al. (2005);
Sohn et al. (2007)
Allen et al. (2004);
Sohn et al. (2007)
CBEL, cellulose-binding elicitor lectin; ETI, effector-triggered immunity; NLP, Nep1-like protein; PAMP, pathogen-associated molecular pattern; PIP1, Phytophthorainhibited protease 1; PTI, PAMP-triggered immunity; ROS, reactive oxygen species; SCR, small cysteine-rich; SERK3/BAK1, SOMATIC EMBRYOGENESIS RECEPTOR
KINASE 3/brassinosteroid (BR)-associated kinase 1.
host cell, to suppress key modulators of the downstream signalling cascade of PTI (Zipfel, 2008).
A number of microbial PAMPs known to elicit a defence
response in plants have been characterized, including bacterial
flagellin, cold-shock proteins, lipopolysaccharides (LPS), elongation factor Tu (EF-Tu), fungal chitin, b-glucans and ergosterol
(Ingle et al., 2006; Nürnberger and Lipka, 2005; Nürnberger
et al., 2004). Indeed, recognized patterns can be narrowed down
to particular conserved motifs or domains within these molecules, such as the 22-amino-acid peptide flg22 of bacterial
flagellin (Felix et al., 1999) and the RNA-binding motif RNP-1 of
bacterial cold-shock proteins (Felix and Boller, 2003). However,
not all PAMPs are recognized by all plant species; for example,
only members of the Brassicaceae respond to EF-Tu (Felix and
Boller, 2003). PAMPs have been defined as surface-exposed,
abundant structures that are common to microbial sources, are
not found in potential eukaryotic hosts and are indispensable for
the microbial lifestyle (Medzhitov and Janeway, 1997). Additions
to these definitions (Gijzen and Nürnberger, 2006) include that
PAMPs act outside the plant plasma membrane and comprise
activities that are not heat labile.
The oomycetes form a phylogenetically distinct group of
eukaryotic microorganisms and consequently possess unique,
essential physiological and structural components that function
as PAMPs. Thus far, many oomycete PAMPs and other elicitors
are secreted proteins (Fig. 1). Consequently, this brings them into
the vicinity of plant cell surface receptors that can trigger PTI.
Some oomycete PAMPs share the characteristics of enzymes or
protein toxins (Gijzen and Nürnberger, 2006). In addition to
these proteinaceous PAMPs and elicitors, it has long been known
that surface-exposed oomycete glucans also trigger defences in
host plants (Sharp et al., 1984). Indeed, a glucan receptor in the
plasma membrane of soybean root cells has been characterized
(Umemoto et al., 1997). In this review, however, we focus on
oomycete proteins that act as PAMPs and elicitors (Table 1), all
of which activate defence responses in the host plants infected
by these oomycetes.
One of the first proteinaceous oomycete PAMPs to be identified is an abundant, calcium-dependent transglutaminase
(TGase) named GP42 that functions in the cell wall of P. sojae
(Brunner et al., 2002; Nürnberger et al., 1994). TGases are multifunctional enzymes vital for many physiological processes,
such as cell differentiation and tissue regeneration (Langston
et al., 2007). The acyl transferase activity of the enzyme performs various in vivo and in vitro protein cross-linking and
modification functions (Brunner et al., 2002). The PAMP-like
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activity of GP42 can be narrowed to Pep-13, a specific
13-amino-acid, high-affinity pattern that has been shown to be
necessary and sufficient to stimulate PTI responses in parsley
cells, including ion effluxes, ROS generation, defence-related
gene activation and phytoalexin synthesis (Brunner et al., 2002;
Nürnberger et al., 1994) (Table 1). TGases with highly conserved
Pep-13 motifs have been identified in all Phytophthora species
investigated (Brunner et al., 2002). Mutational analysis of
Pep-13 revealed that the amino acids important for plant
defence-eliciting activity are also essential for TGase activity,
supporting the concept that plants have evolved PRRs that recognize genus-specific ‘epitope-like’ motifs that are essential for
the function of pathogen-derived molecules (Brunner et al.,
2002).
A further oomycete-specific cell wall PAMP is the Phytophthora cellulose-binding elicitor lectin (CBEL) isolated from P.
parasitica var. nicotianae and found to be a potent elicitor of
necrosis and PTI (Mateos et al., 1997) (Table 1). Thus far, 42
CBEL-like domains have been identified in 28 putative proteins
of P. infestans, P. sojae, P. ramorum and P. parasitica, suggesting that this domain is widely distributed in oomycetes (TortoAlalibo et al., 2005). Like other known PAMPS, an essential
motif sufficient to stimulate defence responses in plants was
pinpointed to two cellulose-binding domains (CBDs) within
CBEL. CBDs belong to the carbohydrate-binding module 1
family, which is almost exclusively found in fungi (Gaulin et al.,
2006). Unexpectedly, although CBEL contributes to adhesion,
the virulence of transgenic strains of P. parasitica suppressed in
CBEL expression was not seriously affected. In spite of this, the
lack of CBEL correlated with the abnormal formation of
papillae-like cell wall thickenings in Phytophthora, suggestive of
a developmental function supporting its classification as a
PAMP (Gaulin et al., 2002). Further studies mutating conserved
aromatic residues in CBEL significantly reduced cellulose
binding. Single mutations in either CBD abolished elicitor activity, whereas mutations in both CBDs were required to eliminate
cell wall binding (Gaulin et al., 2006). The CBDs have no detectable hydrolytic activity and may be perceived by the plant via
the local destabilization of cellulose caused by binding (Dumas
et al., 2008). Supporting research using protoplasts has shown
that the cell wall is necessary for CBEL to elicit a transient
variation of cytosolic calcium levels in tobacco cells. Surprisingly, the cev Arabidopsis mutant, deficient in cellulose synthesis, was more resistant to cell wall-penetrating powdery mildew
fungal pathogens. However, cev mutants constitutively express
jasmonate- and ethylene-dependent defence pathways, suggestive of a surveillance mechanism functioning in the plant cell
wall (Ellis and Turner, 2001).
Insights into the nature of effectors likely to be important for
zoospore attachment have come from studies such as the
cloning of the PcVsv1 gene encoding the ventral vesicle adhesive
in Phytophthora cinnamomi. PcVsv1 contains 47 copies of the
thrombospondin type I repeat motif, a conserved 50-amino-acid
sequence found in a range of extracellular adhesive molecules
secreted by mammalian cells and malarial parasites (Robold and
Hardham, 2005). Its occurrence in the soil-borne Phytophthora
and Pythium species, as well as in representatives of the downy
mildews and white rusts, suggests that it is widely conserved in
the oomycetes. As was the case for CBEL (above), PcVsv1 is
proposed to function in adhesion to plant cell walls (Hardham,
2007). It would be interesting to determine whether there are
examples of plant species that have evolved to recognize PcVsv1
as a PAMP, as its cross-species conservation suggests it would be
a likely candidate.
A number of small cysteine-rich (SCR) proteins that elicit
necrosis and defence responses in plants are secreted by
oomycetes (Kamoun, 2006). In addition to elicitins (below), SCR
proteins include PcF from Phytophthora cactorum, which induces
necrosis and may act as a phytotoxin (Table 1) (Orosomando
et al., 2001). SCR74 and SCR91 are two PcF-like secreted proteins from P. infestans. The Scr74 gene family is induced during
infection, and has been subject to diversifying selection. It
appears that scr74 has undergone gene duplication and recombination, followed by functional divergence that is reminiscent of
molecular co-evolution with the host, suggesting that this family
of proteins could be targets for detection by host surveillance
systems (Liu et al., 2005).
Elicitins are conserved 10-kDa proteins that are secreted by
most species of Phytophthora and Pythium and that elicit a wide
range of defence responses in plants, including the hypersensitive response (HR), a form of programmed cell death (PCD), in
Nicotiana spp. (Kamoun, 2006) (Table 1). Class I elicitins can
bind sterols, such as ergosterol, and function as sterol-carrier
proteins (Vauthrin et al., 1999). This is of potential importance
because Phytophthora spp. (and oomycetes in general) lack the
squalene epoxidase and the 14a-demethylase enzymes required
to convert sterol precursors. Consequently, they cannot synthesize sterols and must acquire them from external sources (Tyler
et al., 2006). Sterol loading has been implicated in elicitin
binding to a plasma membrane receptor in tobacco and in activating the HR (Osman et al., 2001). HR triggered by the elicitin
INF1 from P. infestans has been shown to require the respiratory
burst oxidase Nbrboh (Yoshioka et al., 2003), heat-shock proteins HSP70 and HSP90 (Kanzaki et al., 2003), the ubiquitin
ligase-associated protein NbSGT1 (Peart et al., 2002) and a
MAPK kinase (Takahashi et al., 2007). Recently, a lectin-like
receptor kinase, NbLRK1, has been shown to interact with INF1.
Virus-induced gene silencing of NbLRK1 delayed INF1-mediated
HR, suggesting that it is a component of the protein complex
responsible for INF1 perception and signal transduction
(Kanzaki et al., 2008). Support for the view that INF1 elicitin can
be regarded as a PAMP comes from the observation that ROS
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generation, following INF1 perception, is dependent on the
leucine-rich repeat (LRR) receptor kinase, SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3/brassinosteroid (BR)associated kinase (BAK)1. SERK3/BAK1 has been implicated in
the integration of diverse PAMP perception events into a
general PTI response by forming complexes with PAMP receptors (Heese et al., 2007). It would thus be interesting to determine whether the Nicotiana benthamiana SERK3/BAK1 protein
complexes with NbLRK1.
The cell wall-associated, necrosis-inducing, Phytophthora
protein 1 (NPP1) of P. parasitica induces similar PTI responses
to Pep-13, but also induces cell death (Fellbrich et al., 2002;
Qutob et al., 2006) (Table 1). NPP1-mediated plant defences,
including the oxidative burst, accumulation of pathogenesisrelated (PR) proteins, callose deposition and necrosis, did not
utilize the Pep-13 receptor, suggestive of a converging PTI signalling cascade with other PAMP receptors (Fellbrich et al.,
2002; Qutob et al., 2006). NPP1 is a member of a large class of
proteins, known as Nep1-like proteins (NLPs), present in predominantly plant pathogenic oomycetes, fungi, Gram-negative
and Gram-positive bacteria (Gijzen and Nürnberger, 2006).
Nep1 is a 24-kDa protein that induces necrosis and ethylene
production in plants, initially identified from the culture filtrate
of the cereal pathogen Fusarium oxysporum (Bailey, 1995).
Further analyses of NLP sequences have indicated that an
ancient and fundamental bifurcation occurred during their evolution. Type I NLP domains, with two conserved cysteines, are
found in fungi, oomycetes and bacteria, whereas the Type II
NLP domains, with four conserved cysteines, are not found in
oomycetes (Gijzen and Nürnberger, 2006). Currently, there is
debate about whether NLPs are PAMPs, as there are significant
characteristic differences when compared with Pep-13 and
flg22 responses (Gijzen and Nürnberger, 2006; Qutob et al.,
2006). NLPs can be classed as PAMPs on the basis of the rapid
triggering of plant defence responses associated with other
PAMPs, and because they act outside of the plant cell membrane. However, unlike Pep-13 or flg22, no defined, conserved
amino acid motif in NLPs has been shown to be required
for PTI.
Finally, the crinkling and necrosis (Crn) gene family has been
shown to induce necrosis when expressed inside plant cells
(Kamoun, 2007; Torto et al., 2003). Crn genes are numerous in
oomycete genomes. They encode modular proteins with a highly
conserved N-terminus, followed by highly variable combinations
of domains (Win et al., 2006). The conserved N-terminal domain,
following a signal peptide for secretion, contains a motif,
LXLFLAK, which has been postulated to act in the translocation
of these proteins to the inside of host cells (Kamoun, 2007).
Future work will reveal whether CRN proteins act, like RXLR
proteins (see next section), as cytoplasmic effectors that manipulate host defence or metabolic functions.
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OOMYCETE ETS
Plant pathogenic oomycetes must be capable of overcoming
preformed and inducible defences in at least one host plant
species. Physical barriers, such as the cell wall, cuticle and epicular waxes, successfully thwart infection by non-pathogenic
oomycetes (Attard et al., 2008). Plant pathogenic oomycetes
have evolved specialized infection strategies that facilitate the
invasion of plant cells, including the development of appressoria
for host cell penetration, and the secretion of pectinases and
glucanases that weaken the cell wall, together allowing physical
barriers to be overcome (Latijnhouwers et al., 2003). Oomycetes
must invade plant tissues in such a way that they acquire sufficient nutrients to sustain themselves in the dynamic and potentially hostile environment of the host. Some effectors may thus
serve structural roles, for example, in the extrahaustorial matrix
(Schulze-Lefert and Panstruga, 2003). Others may promote
pathogen dispersal or nutrient leakage, perhaps by modulating
the transition from biotrophy to necrotrophy in the case of hemibiotrophs, such as P. infestans. Indeed, some of the PAMPs and
elicitors referred to above have been proposed to play roles
important for the infection process, other than to suppress host
defences. Thus, CBEL has been implicated in host cell adhesion,
elicitins in the assimilation of sterols, and NLPs, inducing necrosis
in resistant and susceptible plants, may be important in facilitating the transition to the necrotrophic phase of plant colonization (Qutob et al., 2002). A role for NLPs in virulence is
supported by the finding that a knock-out of the NLP1 homologue in the soft-rot enterobacterial pathogen Pectobacterium
atrosepticum results in reduced virulence on potato (Pemberton
et al., 2005).
Recent research has provided the first examples of oomycete
effectors that suppress PTI and thus overcome the initial hurdle
in establishing a successful infection (Fig. 1). Two broad classes
of effectors can be identified: those that act extracellularly to
suppress secreted defence-related proteins, and those that act
intracellularly, presumably to suppress the defence-related
signal transduction, regulation and trafficking that lead to PTI
or ETI.
Extracellular oomycete effectors
Several plant PR proteins, such as glucanases, chitinases and
proteases, are secreted hydrolytic enzymes. From a host viewpoint, such enzymatic activities represent potentially important
defence mechanisms through both direct degradation of the
pathogen mycelial wall and, in the case of glucanases, indirectly
via the release of b-1,3-glucan oligosaccharide elicitors (van
Loon et al., 2006). Fungal and bacterial plant pathogens have
evolved diverse mechanisms for protection against the activities
of these PR proteins (Abramovitch and Martin, 2004; Chisholm
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et al., 2006). Similarly, plant pathogenic oomycetes, such as Phytophthora, have also evolved mechanisms to escape the enzymatic activity of secreted hydrolases. Oomycetes contain little
chitin in their cell wall and are therefore resistant to plant
chitinases (Bartinicki-Garcia and Wang, 1983). In addition, they
have evolved active counter-defence mechanisms by secreting
inhibitory proteins that target host glucanases and proteases
(Table 1). The glucanase inhibitor proteins GIP1 and GIP2,
secreted by P. sojae, inhibit the soybean endo-b-1,3-glucanase
EgaseA (Rose et al., 2002). Four genes with similarity to P. sojae
GIPs have been identified in the P. infestans genome. Analyses of
P. infestans-inoculated tomato leaves showed that P. infestans
GIPs and tomato EGases are present in the apoplast and form
stable complexes in planta. Codon evolution analyses of GIP
homologues identified several positively selected peptide sites,
and structural modelling has revealed that they are in close
proximity to rapidly evolving EGase residues, suggesting that the
interaction between GIPs and EGases has the hallmarks of tight
molecular co-evolution (Damasceno et al., 2008), the principles
of which are described in more detail below for the molecular
co-evolution between effector and R genes.
In addition, 18 extracellular protease inhibitor genes have
been identified in P. infestans that belong to two major structural classes: (i) Kazal-like serine protease inhibitors (called
EPI1–14); and (ii) cystatin-like cysteine protease inhibitors
(EPIC1–4). EPI1 and EPI10 interact with and inhibit a secreted
subtilisin-like serine protease of tomato, the PR protein P69B
(Tian et al., 2004, 2005). EPIC1 and EPIC2B have been shown
to be unstable in tomato apoplastic fluids and to be degraded
by tomato P69B. However, EPI1 protects both proteins from
degradation. Affinity-purified P69B was sufficient to degrade
EPIC1 and EPIC2B, but not EPI1, suggesting selectivity in
degradation by P69B. Co-immunoprecipitation experiments
revealed that EPIC2 interacts with a novel papain-like extracellular cysteine protease, termed Phytophthora-inhibited protease 1 (PIP1). The interaction was further confirmed by
co-immunoprecipitation using in planta-expressed PIP1 protein.
Together, these findings suggest that a cascade of inhibition of
host proteases initiated by EPI1 occurs in the tomato apoplast
during infection by P. infestans. In addition, this study provides
biochemical evidence for a direct contribution of the PR protein
P69B subtilase to defence through the degradation of proteins
from invading pathogens (Tian et al., 2007). Like GIPs, PIP1 has
recently been shown to be under strong diversifying selection.
In addition, it is a target of the Cladosporium fulvum avirulence protein AVR2, which acts as a protease inhibitor (Shabab
et al., 2008). More recently, RCR3, a tomato protease targeted
by the C. fulvum AVR2 protein, has also been shown to be
targeted by EPIC1 and EPIC2B from P. infestans, demonstrating
that unrelated pathogens manipulate the same defence targets
in the host to establish infection (Song et al., 2009). The secre-
tion of extracellular serine protease inhibitors seems likely to
be part of a common infection strategy for Phytophthora spp.,
because P. parasitica expresses a gene similar to epi1 (Panabieres et al., 2005) and secreted Kazal-like proteins form a
diverse family of at least 35 members from five plant pathogenic oomycetes, including the downy mildew Plasmopara
halstedi (Kamoun, 2006).
Intracellular oomycete effectors
The biochemical activities of intracellular oomycete effectors are
poorly understood. However, we might expect some common
features with the biochemical functions of bacterial pathogen
T3SS effectors that often mimic or inhibit eukaryotic cellular
functions on delivery into the host cell (Block et al., 2008; Grant
et al., 2006). In recent years, six oomycete cytoplasmic effectors
have been discovered through their avirulence (AVR) activity,
triggering an HR on host genotypes with corresponding disease
resistance (R) genes (Allen et al., 2004; Armstrong et al., 2005;
van Poppel et al., 2008; Rehmany et al., 2005; Shan et al., 2004;
Vleeshouwers et al., 2008) (Table 2). All six AVR proteins
contain, within the N-terminal 60 amino acids, a secretory signal
peptide and a conserved domain featuring the motif RXLR, followed by a high frequency of acidic (D/E) residues, and a
C-terminal domain(s) associated with virulence function (Birch
et al., 2006, 2008; Win et al., 2007). The oomycete RXLR motif is
similar in sequence and position to the host cell targeting signal
(HT/Pexel motif) required for the translocation of proteins from
malaria parasites (Plasmodium spp.) into host red blood cells
(Hiller et al., 2004; Marti et al., 2004). Indeed, the oomycete
RXLR signal has been demonstrated to function for effector
delivery in the malarial system (Bhattacharjee et al., 2006).
Whisson et al. (2007) and Dou et al. (2008a) have since shown
that the RXLR motif is required for the translocation of the P.
infestans effector AVR3a and the P. sojae effector Avr1b, respectively, inside host plant cells. Phytophthora infestans transformants expressing AVR3a, in which the RXLR and EER regions
were replaced, singly or in combination, with alanine residues,
or with alternative amino acids that conserve the physicochemical properties of the protein, failed to deliver AVR3a from the
extrahaustorial matrix to the inside of the plant cell (Whisson
et al., 2007). Translocation was confirmed using the Escherichia
coli GusA gene fused to the signal peptide (SP)-RXLR-EERencoding domains of AVR3a. b-Glucuronidase (GUS) activity
was observed in plant cells in contact with the haustoria of P.
infestans transformants expressing the SP-RXLR-EER::GusA
fusion, but not with transformants expressing a version in which
the motifs were replaced with alanine residues (SP-AAAA-AAAGUS) (Whisson et al., 2007).
Dou et al. (2008a) have demonstrated, using co-bombardment and a purified Avr1b::GFP fusion protein, that the RXLR
© 2009 THE AUTHORS
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Table 2 Cloned resistance (R) genes and avirulence (Avr) genes from oomycete–plant interactions.
Cloned R gene
Source
Reference
Resistance to
Cognate Avr gene
Reference
R1
R3a
RB/Rpi-Blb1
Solanum demissum
S. demissum
S. bulbocastanum
Phytophthora infestans
P. infestans
P. infestans
Avr3a
AVR-blb1(IpiO)
Armstrong et al. (2005)
Vleeshouwers et al. (2008)
Rpi-sto1
Rpi-pta1
Rpi-Blb2
Unknown
RPP1 (-WsC, -WsA,
-WsB)
RPP2 (RPP2A&B)
RPP4
RPP5
RPP7
RPP8
RPP13 (-Nd, -RLD)
Dm3
Unknown
S. stoloniferum
S. papita
S. bulbocastanum
S. demissum
Arabidopsis thaliana
Ballvora et al. (2002)
Huang et al. (2005)
Song et al. (2003);
van der Vossen et al. (2003)
Vleeshouwers et al. (2008)
Wang et al. (2008)
van der Vossen et al. (2005)
Avr4
ATR1
van Poppel et al. (2008)
Rehmany et al. (2005)
ATR13
Allen et al. (2004)
AVR1b-1
Shan et al. (2004)
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
A. thaliana
Lactuca sativa
Glycine max
Botella et al. (1998)
Sinapidou et al. (2004)
van der Biezen et al. (2002)
Parker et al. (1997)
Eulgem et al. (2007)
McDowell et al. (1998)
Bittner-Eddy et al. (2000)
Shen et al. (2002)
and EER motifs in Avr1b are required for translocation across the
host plasma membrane, and that such translocation is independent of the pathogen. They showed that the amino acids R at
position 1 and L at position 3 were critical for translocation by
the RXLR motif. Dou et al. (2008a) and Grouffaud et al. (2008)
have both shown that the Plasmodium HT/Pexel motif will function in Phytophthora for effector delivery into plant cells. This
indicates an ancient, conserved means of effector delivery into
plant and animal cells that has been exploited, possibly through
convergent evolution, by these distantly related pathogens.
The secreted RXLR complement of an oomycete can be considered analogous to the T3SS repertoire of a bacterial plant
pathogen, and the discovery of the effector functions of the large
number of secreted RXLR-containing proteins found in oomycete
genome sequences will provide the focus for much future
research in the field of oomycete plant pathology. Already, some
insights into RXLR effector virulence functions have been published (Table 1). The P. infestans RXLR effector Avr3a was identified by association genetics, and it was shown that avirulent
and virulent alleles differ at only two amino acid positions, K80E
and I103M, respectively, in the C-terminal domain (Armstrong
et al., 2005). AVR3aK80I103, in addition to being recognized by
R3a, is able to suppress hypersensitive cell death induced by the
P. infestans elicitin, INF1, pointing to a possible virulence function. The AVR3aE80M103 form, in contrast, is not recognized by
R3a or able to suppress INF-mediated cell death (Bos et al.,
2006). Cell death suppression activity is a common feature of
bacterial effectors, and the observation that RXLRs have this
ability in plants is entirely consistent with expected models of
effector function. In addition to AVR3a, the P. sojae RXLR effector Avr1b can suppress PCD triggered by the mouse BAX protein
in yeast, soybean (Glycine max) and N. benthamiana cells (Dou
P. infestans
P. infestans
Hyaloperonospora
arabidopsidis
H. arabidopsidis
H. arabidopsidis
H. arabidopsidis
H. arabidopsidis
H. arabidopsidis
H. arabidopsidis
Bremia lactucae
P. sojae
et al., 2008b). Computational work has identified conserved
motifs within the C-terminal domains of Avr1b, and many other
effectors, that have been termed W, Y, K and L, and that can occur
in multiple copies (Dou et al., 2008b; Jiang et al., 2008). PCD
suppression by Avr1b is dependent on the W and Y motifs. Two
additional effectors from P. sojae (PsAvh331 and PsAvh163) and
one from H. arabidopsidis (HpRxL96), all encoding proteins containing multiple W and Y motifs, are also able to suppress PCD
(Dou et al., 2008b).
Two additional RXLR proteins have also been assigned virulence functions (Table 1). Alleles of ATR1 and ATR13 from H.
arabidopsidis confer enhanced virulence to Pseudomonas syringae pathovar tomato DC3000 on susceptible Arabidopsis accessions, suggesting that ATR1 and ATR13 positively contribute to
pathogen virulence inside host cells. In addition, ATR13 alleles
have been shown to suppress bacterial PAMP-triggered callose
deposition in susceptible Arabidopsis. Furthermore, expression of
another allele of ATR13 in plant cells suppressed PAMPtriggered ROS production in addition to callose deposition
(Sohn et al., 2007).
ETI AND BEYOND
Effectors deployed by invading pathogens to, for example, gain
access to host cell nutrients and/or suppress host defences (ETS),
simultaneously provide the plant with new targets for detection.
In the next layer of defence, plants utilize R proteins in a surveillance system to reveal effector (AVR) proteins and activate ETI
(Jones and Dangl, 2006) (Fig. 1). This response involves the HR,
which has been associated with almost all forms of resistance
towards oomycetes, including non-host resistance (Kamoun
et al., 1999).
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I. HEIN et al.
The traditional view of R gene perception of an Avr gene
follows the gene-for-gene concept that suggests a direct interaction between both protein products, similar to the receptor–
ligand relationship of antibodies and antigens (Flor, 1971). In the
majority of cases, however, recognition of an AVR protein by a
cognate R protein may be indirect, involving activation of the R
protein on recognition of effector activity on virulence targets,
also know as ‘pathogen-induced modified self-molecular patterns’, previously described in the ‘Guard’ hypothesis (van der
Biezen and Jones, 1998; Dangl and Jones, 2001). With the ability
to initiate PCD, R protein activation is tightly regulated. The
largest class of R proteins comprises nucleotide-binding site
(NBS) and LRR domains, and current models postulate that many
form multiprotein ‘trigger’ complexes that, by intra- and intermolecular interactions, maintain the R proteins in an inactive
form until effectors are detected (Belkhadir et al., 2004; Friedman and Baker, 2007). The LRR domain might play a dual role,
acting both as a recognition specificity determinant and as a
repressor that prevents inappropriate NBS activation (Belkhadir
et al., 2004). Evolutionary analysis of R genes and their structure
has demonstrated selection pressure on the LRR-encoding
regions, and also on other domains within R genes which, in light
of the dual function, may point towards the co-evolution of
domains to establish or maintain the guarding function on the
virulence target(s) whilst maintaining the inhibitory function
(Belkhadir et al., 2004; McDowell and Simon, 2006). This scenario sets the scene for a very close evolutionary contest on a
molecular level between pathogen effectors to evade detection
and plant R proteins and virulence targets to retain recognition.
It has been speculated that plant R proteins may guard key,
general pathogen targets, new pathogen-specific virulence
targets, and deploy decoys to maximize the chance of pathogen
recognition, all of which could then be subject to evolutionary
pressure (Friedman and Baker, 2007; van der Hoorn and Kamoun,
2008; McDowell and Simon, 2006).
Analysis of the fully sequenced model plant Arabidopsis
thaliana ecotype Columbia (Col-0) has revealed approximately
149 genes that encode NBS–LRR-containing R proteins (Meyers
et al., 2003). This class can be further divided according to the
N-terminal domain: either a Toll/interleukin-1 family of receptor
(TIR) domain, or a coiled-coiled (CC) region (Meyers et al., 1999).
Interestingly, about two-thirds of NBS–LRR genes are found in
clusters (Meyers et al., 2003). This arrangement is thought to
reflect the evolution of R genes by a process relying on duplication, subsequent diversification and adaptive selection, also
known as birth-and-death (Lehmann, 2002; Michelmore and
Meyers, 1998; Nei et al., 1997). R genes can be under either
purifying selection, acting against gene variation to maintain R
protein function, or diversifying selection, which provides a
means for new R gene recognition specificities. The latter is
characterized by a rate of non-synonymous nucleotide substitu-
tions (KA), resulting in amino acid changes, that is greater than
the rate of synonymous nucleotide substitutions (KS), which do
not alter the amino acid (KA : KS ratio > 1). In contrast, purifying
selection typically displays a KA : KS ratio < 1.
Evolution of the RGC2 locus in cultivated lettuce and wild
relatives has shown that different selective forces act on R genes.
Multiple mechanisms, including recombination, point mutations
and insertions/deletions, drive R gene evolution, and different
genes within a cluster have the potential to evolve independently
(Kuang et al., 2004, 2005). The RGC2 locus comprises a large
cluster of R genes, and a gene for resistance towards root aphid
has been mapped alongside at least 10 Dm genes that confer
resistance towards the oomycete downy mildew pathogen
Bremia lactucae (Meyers et al., 1998a, b). Within this one cluster,
two types of RGC2 gene have been identified that reflect a
heterogeneous rate of evolution, yielding fast-evolving ‘type I’
genes, characterized by frequent sequence exchanges associated
with chimeric genes and haplotype diversity, including variable
gene copy numbers, and more slowly evolving ‘type II’ genes, that
infrequently exchange sequences and maintain a higher similarity
between orthologues than paralogues. In line with type I genes,
the RGC2 copy number within seven genotypes from three
species ranged from 12 in cv. Calmar to 32 in cv. Diana (Kuang
et al., 2004). Furthermore, gene variation studies focusing on the
LRR region of three Arabidopsis genes have shown that some,
including the hypervariable RPP13 gene effective against the
oomycete H. arabidopsidis, exhibit levels of diversity within populations that are similar to the diversity observed between populations, and could thus provide a means to maintain a high level
of polymorphism (Ding et al., 2007).
Regardless of the mechanism of R gene diversification, in
terms of plant–pathogen co-evolution, two extreme outcomes
are conceivable: (i) balancing selection, resulting in long-lived
and high allelic diversity of R genes by maintaining stable polymorphisms (Bergelson et al., 2001), previously described as
‘trench warfare’ (Stahl et al., 1999); and (ii) selective sweeps,
resulting in young R genes and monomorphic R gene loci as a
consequence of the transient maintenance of polymorphism and
the replacement of old R genes by new variants. This has been
described as an ‘arms race’ (Bergelson et al., 2001). Interestingly,
a genome-wide survey of polymorphisms within the LRR region
of 27 representative R genes in 96 Arabidopsis thaliana accessions has found that both extremes are not common, and that
R gene co-evolution has resulted in a continuum of possible
states between ‘trench warfare’ and an ‘arms race’ (Bakker
et al., 2006).
Cloned and characterized plant R genes conferring resistance
to oomycetes, and oomycete Avr effectors, are shown in Table 2.
To date, all oomycete AVR proteins are RXLR effectors, and
computational analysis of the genome sequences of P. sojae, P.
ramorum, P. infestans and H. arabidopsidis has predicted hun-
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Zig-zag-zig in oomycete–plant interactions
dreds of candidate RXLR genes in each oomycete (Birch et al.,
2008; Jiang et al., 2008; Jones and Dangl, 2006; Whisson et al.,
2007; Win et al., 2007), which is in stark contrast with the small
repertoire of approximately 30 T3SS effectors required for bacterial infection (Jones and Dangl, 2006). Furthermore, although only
few RXLR-containing effectors appear to be sufficiently conserved
between sequenced oomycetes to have originated from a
common ancestor, it is apparent that many effectors within
species contain paralogous family members (Win et al., 2007).
Taken together, these observations suggest an extraordinary
potential for virulence function redundancy that may be governed
by family members and/or evolutionarily unrelated effector
sequences (Birch et al., 2008). In the light of this, it is of little
surprise that the oomycetes comprise such formidable pathogens.
Win et al. (2007) determined the ratio between nonsynonymous and synonymous nucleotide substitutions among
RXLR gene family members in P. sojae, P. ramorum and H.
arabidopsidis, and demonstrated that a great proportion of gene
families are under positive selection. Furthermore, the C-terminal
regions of effectors, mainly associated with virulence function,
displayed a significantly higher rate of positive selection than did
the N-terminal regions, which contain domains implicated in
effector secretion and host targeting (Win et al., 2007). Reminiscent of the genomic organization of R genes, many RXLR effectors under positive selection were found in clusters, which has
been postulated to be an important mechanism of gene duplication and diversification, and is consistent with their roles as
modulators and targets of host resistance (Win et al., 2007).
Pathogen responses to effector detection can vary greatly and
may be dependent on the level of functional redundancy within
the pathogen. So far, and discussed below, evidence indicates
that oomycetes can (i) lose effectors without loss of virulence,
pointing towards redundancy in virulence function; (ii) diversify
effectors to maintain virulence function and simultaneously
evade recognition; and (iii) utilize so far unidentified inhibitors
that act downstream of the recognition of avirulence genes to
prohibit resistance responses.
The effector genes Avr4 from P. infestans and Avr1b from P.
sojae provide evidence for functional redundancy by the
approaches used to evade recognition. Avr4 is recognized by the
uncharacterized potato R4 gene. Phytophthora infestans isolates
that are virulent on R4-containing plants possess truncated and
non-functional avr4 alleles, indicating that the loss of an effector
has no detrimental effect on overall pathogen virulence (van
Poppel et al., 2008). Avr1b, recognized as an Avr gene in
soybean plants carrying the cognate Rps1b resistance gene, was
identified by map-based cloning. All analysed P. sojae isolates
virulent on Rps1b plants contain Avr1b, but either with numerous amino acid substitutions or, in other isolates, fail to accumulate Avr1b mRNA and thus avoid recognition without obvious
consequences to pathogen fitness (Shan et al., 2004). Neverthe-
555
less, P. sojae transformants over-expressing Avr1b displayed
increased virulence compared with control transformants (Dou
et al., 2008b). Interestingly, Avr1b and the paralogous Avh1b
protein exhibit sequence similarity to AVR3a from P. infestans,
which triggers R3a-dependent PCD (Armstrong et al., 2005; Win
et al., 2007). Indeed, Avh1b, but not Avr1b, is weakly recognized
by R3a. Comparison with an Avr1b variant, Avr1bAAM20939, that
also evades recognition by R3a, highlighted nine amino acids
under positive selection that differ between the two Avr1b variants and Avh1b. Mutagenesis of an aromatic threonine from
Avh1b into a basic lysine, as found in Avr1b-1, abolished the
R3a-dependent HR eliciting activity (Win et al., 2007). Future
work might determine whether Avh1b and Avr3a have similar
functions and seek out similar virulence targets, or whether the
elusive Rps1b gene and R3a guard similar or different virulence
targets.
In addition to the virulent or avirulent allele of Avr3a, the
Avr3a locus contains two paralogues, referred to as PEX147-2
and PEX147-3. Phylogenetically, the two alleles display a closer
relationship compared with the paralogues, which suggests that
duplication and divergence under positive selection are the
driving mechanisms to avoid detection in this locus (Armstrong
et al., 2005). The R3a gene from Solanum demissum, located
within the R3 locus on chromosome 11, was cloned by comparative genomics and has revealed homology and synteny to the I2
locus in tomato that mediates resistance towards the fungus
F. oxysporum f. sp. lycopersici (Huang et al., 2005). This locus
appears to be an R gene hotspot in the Solanaceae and contains,
in addition to R3a, other P. infestans resistance genes, such as
the closely linked R3b (Huang et al., 2004) and, potentially,
R5-R11 (Huang, 2005), as well as quantitative resistance
towards the cyst nematode Globodera rostochiensis in potato, R
genes effective towards Stemphylium spp. and to yellow leaf curl
virus in tomato, and resistance towards tobacco mosaic virus in
pepper (Huang et al., 2005). Sequence analysis at both loci suggests that these R genes share a common ancestral gene, but has
also shown that, in potato, the locus has not only expanded
significantly in size, but also in the number of R genes. Within
the R3a cluster, four paralogues were identified, all of which
are constitutively expressed and show hallmarks of diversifying selection. R3a haplotype comparison between other
S. demissum, as well as evidence of sequence exchange between
paralogues, is consistent with a type I class of R gene that
evolves rapidly (Huang et al., 2005).
The more durable Rpi-blb2 resistance gene from Solanum
bulbocastanum has been identified within a cluster of R genes
on chromosome 6 and is similar to the Mi-1 gene from tomato
that mediates resistance towards root-knot nematodes, aphids
and white flies (van der Vossen et al., 2005). Overall, two clusters
have been identified at both loci and, similar to the I2 locus,
haplotype analysis of the Mi-1 loci in Solanum peruvianum and
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I. HEIN et al.
S. lycopersicum has shown a comparable structure and organization indicative of a conserved and slow evolution. In contrast,
the same clusters have expanded significantly in S. bulbocastanum and the numbers of homologues in both clusters has
increased, reminiscent of the expansion of the R3 locus (van der
Vossen et al., 2005). It is tempting to speculate that the contrasting evolutionary fates of the slowly evolving I2 and Mi-1 loci
in tomato, and the faster evolving R3a and Rpi-blb2 clusters in
potato, reflect the converse evolutionary potential of the interacting pathogen and/or the cognate effectors, with slower evolving F. oxysporum and root-knot nematodes on the one side and
rapidly changing P. infestans on the other.
A second, also potentially more durable R gene isolated from
chromosome 8 in S. bulbocastanum, RB/Rpi-blb1, has been classified as a slow-evolving type II gene (Song et al., 2003; van der
Vossen et al., 2003). A high level of synonymous nucleotide
substitutions is apparent among paralogues of the Rpi-blb1 haplotype, which indicates synonymous divergence and suggests
that these genes are relatively old and could be subject to
balancing selection (van der Vossen et al., 2003). The cognate
P. infestans Avr gene ipiO is highly expressed at the tips of
invading hyphae (van West et al., 1998), and at least two closely
linked genes ipiO1 and ipiO2 exist as part of the PexRD6 family
of effectors (Pieterse et al., 1994; Vleeshouwers et al., 2008).
Using a candidate gene approach, Vleeshouwers et al. (2008)
identified and cloned functional Rpi-blb1 orthologues from
Solanum stoloniferum (Rpi-sto1) and Solanum papita (Rpipta1), and their function could be demonstrated by transient
co-expression with ipiO in N. benthamiana, yielding, in all cases,
an HR similar to the co-expression of Rpi-blb1 with ipiO. In a
similar study, Rpi-blb1 orthologues were successfully amplified,
not only in S. stoloniferum and S. papita, but also in Solanum
polytrichon, whereas amplification of orthologous genes from
the unrelated Rpi-blb2 gene failed to yield homologous genes
(Wang et al., 2008). Considering the R gene diversity and recognition specificity observed between and within populations, it is
of no surprise that effector recognition between Solanum
species and within species accessions varies significantly, as
shown by Vleeshouwers et al. (2008).
The lettuce gene Dm3, a fast-evolving type I RGC2 gene that
confers resistance to B. lactucae races expressing the avirulence
gene Avr3 (Kuang et al., 2004), was found at a very low frequency in wild lettuce accessions (Kuang et al., 2006). Indeed,
within a comprehensive screen of 1033 accessions from 49
natural populations, only one contained Dm3, which could be
interpreted as either a very recent evolutionary event that led to
Dm3 specificity or deletion of this gene caused by unequal crossing over. The latter was further supported by a high conservation
of the 5′ region of Dm3 in about 30% of accessions and the
detection of smaller Dm3-specific sequence fragments in over
40% of accessions. This study also indicated a high level of
diversity within the Dm LRR domains in these wild populations,
which could indicate a high level of Dm gene variation, in line
with at least eight resistance specificities to B. lactucae that
have mapped to the RGC2 locus (Kuang et al., 2006).
The R1 gene for potato resistance to late blight was isolated
from an R gene hotspot on chromosome 5 that contains resistances towards potato virus X (X, Rx2 and Nb) and major quantitative trait loci for resistance to P. infestans, Globodera pallida
(Gpa and Gpa5) and Globodera rostochiensis (Gpr1) (Ballvora
et al., 2002; Kuang et al., 2005). Interestingly, the Rx2 gene is,
similar to R1, a member of the CC-NBS-LRR family of R genes,
but belongs to a different class, as both genes share only 32%
sequence identity. A comparative study of the R1 locus in all
three genomes (haplotypes) of the allohexaploid species S.
demissum has revealed three different types of R gene with
homology to R1, Prf and Bs4, indicative of a complex R gene
cluster (Kuang et al., 2005). In agreement with previous findings,
the evolutionary rates of these different R genes were unequal.
Both Prf-like and Bs4-like potato homologues at the R1 cluster
comprise slow-evolving type II genes, whereas R1 homologues
form three distinct subgroups, display variation in copy numbers,
contain chimeric sequences because of frequent sequence
exchanges reminiscent of fast-evolving type I resistance genes,
and show diversifying selection at the LRR region (Kuang
et al., 2005).
The interaction between Arabidopsis and the oomycete pathogen H. arabidopsidis provides a powerful model system to study
the co-evolution of R genes and cognate Avr genes (Koch and
Slusarenko, 1990). R genes in Arabidopsis that recognize effectors from the oomycete pathogen H. arabidopsidis are referred
to as RPP genes and comprise both CC (e.g. RPP7, 8, 13) and TIR
(e.g. RPP1, 2a, 5) types of R gene (Holub, 2001) (Table 2). Hyaloperonospora arabidopsidis Avr genes are referred to as ATR
(Arabidopsis thaliana recognized) genes. ATR1 and ATR13, both
encoding RXLR effectors, have been well studied (Allen et al.,
2004; Rehmany et al., 2005). A number of RPP genes have been
identified in R gene hotspots and cloned from various ecotypes
(reviewed by Slusarenko and Schlaich, 2003). This review focuses
on two genes, RPP1 and RPP13, as the cognate ATR1 and ATR13
genes from H. arabidopsidis are known.
One clear example for balancing selection has been found for
the single-copy RPP13 gene, the most polymorphic R gene analysed in Arabidopsis, which exhibits an extreme variability and
diversifying selection within the LRR domain (Rose et al., 2004).
A collection of diverse and functionally distinct RPP13 alleles has
been maintained in populations and is mirrored by a balancing
selection of the ATR13 gene, which, in turn, displays high accumulation of non-synonymous polymorphism that is not limited
to alleles that avoid recognition by certain RPP13 alleles (Allen
et al., 2004; Rose et al., 2004). A study of naturally occurring
variation of ATR13 alleles has identified specific amino acid
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Zig-zag-zig in oomycete–plant interactions
positions within a C-terminal domain that are associated with
recognition by RPP13Nd (Niederzenz allele). The study showed
that the N-terminal signal peptide and RXLR regions exhibit
sequence conservation. Indeed, deletion of the N-terminal
domains has no effect on recognition, reminiscent of the recognition of AVR3aK80I103 by R3a (Allen et al., 2008). Mutations in a
conserved leucine/isoleucine repeat within ATR13 alter recognition specificity. However, these changes do not occur in natural
populations, leading the authors to postulate that this domain is
required for virulence (Allen et al., 2008). Domain swap experiments between Rpp13Nd (Niederzenz allele), which recognizes
ATR13Emco5, and RPP13Col, which fails to recognize ATR13Emco5,
have shown that the LRR domain is required for the recognition
and subsequent HR (Rentel et al., 2008).
Candidate R genes for the complex RPP1 loci were first identified from the Arabidopsis accession Wassilewskija, and functional analysis showed that three out of four tightly linked TIRNBS-LRR genes (RPP1-WsA, RPP1-WsB, and RPP1-WsC), with a
distinctive but overlapping recognition specificity, explained the
resistance spectrum previously associated with RPP1, RPP10 and
RPP14 (Botella et al., 1998). The underlying evolutionary mechanism in the generation of this locus could be explained by at
least two tandem duplications, followed by sequence divergence
and positive selection, mainly within the LRR domain, in the form
of non-synonymous amino acid substitutions and the loss of a
complete LRR in RPP1-WsC. Cloning of six diverse ATR1 alleles
from eight H. arabidopsidis isolates has played a crucial role in
unravelling the recognition specificity of RPP1 alleles. Again, the
C-terminal part of the effector determines the recognition and is
under strong diversifying selection (Rehmany et al., 2005). Some
R genes, such as RPP1Nd (Niederzenz allele), recognized a single
allelic form of ATR1NdWsB, whereas RPP1-WsB recognized three
additional highly variable allelic forms, and the RPP1-WsA
cognate ATR gene remains elusive (Rehmany et al., 2005).
ETS2 and beyond?
To avoid recognition, pathogens may ‘shed’ Avr genes, as shown
for the apparently redundant effectors Avr4 from P. infestans
and Avr1b from P. sojae, or enter a tightly entwined
co-evolutionary battle with R genes, as shown above, for
example, for ATR13 and RPP13. However, additional mechanisms are also conceivable. Two independent reports have
shown that, although effector and R gene co-expression yields
an HR, some pathogen isolates that express these effectors
manage to evade recognition on plants carrying the cognate R
gene. This could be explained by additional effectors that are
deployed by the pathogen to suppress recognition or downstream signalling components of ETI. The Arabidopsis R gene
RPP1-WsB from the accession Wassilewskija (see above)
initiates an HR if co-bombarded with the ATR1NdWsB gene from
557
H. arabidopsidis isolate Emco5. However, although ATR1NdWsB is
expressed in Emco5, this isolate does not trigger an HR in the
Arabidopsis accession Ws-0 (Rehmany et al., 2005). Similarly,
bacterial T3SS delivery of the Emco5 form of ATR13 triggers an
HR in Arabidopsis accession Ws-0 (Sohn et al., 2007). A possible
explanation is that Emco5 possesses an additional effector(s)
that suppresses either the recognition of ATR1 and ATR13, or
the downstream signalling or regulation of PCD following
recognition.
CONCLUSIONS
Every step in pathogen invasion provides plants with new opportunities for pathogen recognition or to stop vital virulence components from either entering the plant or leaving pathogen cells.
Similarly, pathogens could potentially target and manipulate
every step in the plant disease resistance response. Consequently, co-evolution may affect a much larger number of plant
and pathogen molecules than is currently known. Recently, the
potential for opposing selective forces for the host virulence
target of pathogen effectors was highlighted (van der Hoorn and
Kamoun, 2008). Indeed, depending on the presence or absence
of guarding R genes, virulence targets could potentially evolve to
enhance effector recognition or to avoid effector manipulation,
whilst maintaining their in planta function. Moreover, virulence
targets could represent decoys that are deployed solely to detect
effectors and are thus free to co-evolve to maintain effector
interactions. Furthermore, a study of the RPP5 loci has shown
that R genes in this cluster can be regulated by RNA silencing
and might thus be able to respond to pathogens that perturb
RNA silencing (Yi and Richards, 2007).
The genomics era is providing us with both oomycete and
host genome sequences which act as blueprints for the effectors
that are required to suppress or otherwise manipulate plant
responses to a wide range of conserved or rapidly evolving
molecules. The scene is set for an expansion in genome sequencing, revealing the sequence diversity in PAMP, elicitor and effector proteins within and between oomycete species that reveals
the complex molecular co-evolutionary battles between pathogen and host to establish or prevent disease.
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