Host-pathogen warfare at the plant cell wall.

Abstract

Plants have evolved sensory mechanisms to detect pathogen attack and trigger signalling pathways that induce rapid defence responses. These mechanisms include not only direct detection of pathogen-derived elicitors (e.g. pathogen-associated molecular patterns (PAMPs) and avirulence factors or effectors) but also indirect sensing of pathogens' impact on the host plant. Among the first plant barriers to pathogen ingress are the cell wall and the cuticle. For those pathogens that penetrate the plant cell wall to gain access to water and nutrients of the plant protoplast, small wounds at penetration sites are created by enzymatic or physical disruption of the plant cell wall. Thus, cell wall integrity sensing is one mechanism by which plants may detect pathogen attack. Some plant cell wall fragments, notably oligogalacturonic acids, elicit similar defence responses in plants as the non-specific PAMP elicitors (e.g. production of reactive oxygen species, elevated expression of defence-associated genes), suggesting that PAMP signalling may provide a good model for studying cell wall integrity sensing in plants. However, much remains to be discovered about this sensing mechanism.

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Host–pathogen warfare at the plant cell wall
Kian He ´maty, Candice Cherk and Shauna Somerville
Plants have evolved sensory mechanisms to detect pathogen
attack and trigger signalling pathways that induce rapid
defence responses. These mechanisms include not only direct
detection of pathogen-derived elicitors (e.g. pathogen-
associated molecular patterns (PAMPs) and avirulence factors
or effectors) but also indirect sensing of pathogens’ impact on
the host plant. Among the first plant barriers to pathogen
ingress are the cell wall and the cuticle. For those pathogens
that penetrate the plant cell wall to gain access to water and
nutrients of the plant protoplast, small wounds at penetration
sites are created by enzymatic or physical disruption of the
plant cell wall. Thus, cell wall integrity sensing is one
mechanism by which plants may detect pathogen attack.
Some plant cell wall fragments, notably oligogalacturonic
acids, elicit similar defence responses in plants as the non-
specific PAMP elicitors (e.g. production of reactive oxygen
species, elevated expression of defence-associated genes),
suggesting that PAMP signalling may provide a good model for
studying cell wall integrity sensing in plants. However, much
remains to be discovered about this sensing mechanism.
Address
Energy Biosciences Institute, Calvin Laboratory MC 5230, UC Berkeley,
Berkeley, CA 94720, USA
Corresponding author: Somerville, Shauna (ssomerville@berkeley.edu)
Current Opinion in Plant Biology 2009, 12:406–413
This review comes from a themed issue on
Biotic Interactions
Edited by Xinnian Dong and Regine Kahmann
Available online 16th July 2009
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.06.007
Introduction
The plant cell wall is an exoskeleton surrounding the cell
protoplast, and is composed of a highly integrated and
structurally complex network of polysaccharides, in-
cluding cellulose, hemicelluloses and pectin (for compre-
hensive reviews of the cell wall structuresee [1]). In brief,
cell wall synthesis begins when a pectin rich middle
lamella is deposited at the cell plate during cytokinesis.
Then,theprimarycellwallissynthesizedandremodelled
following cell growth. Finally, a thicker secondary cell
wall is deposited once the cell has reached its final
size. Growth and cell shape are largely determined by
the balance between expansion driven by turgor and
constraint provided by the plant cell wall. The cell wall
is also a highly dynamic structure that is constantly
remodelled during growth and development and in
response to environmental cues. For example, upon
pathogen attack, plants often deposit callose rich cell
wall appositions (i.e. papillae) at sites of attempted
pathogen penetration, accumulate phenolic compounds
and various toxins in the wall and synthesize lignin-like
polymers to reinforce the wall [2?]. Thus, much like the
ramparts or fortress walls of ancient cities, the plant cell
wall can be an important defensive structure that many
pathogens encounter first before confronting intracellular
plant defences [3,4]. Pathogens use mechanical force or
release cell wall degrading enzymes to break down this
barrier. At the cell wall, they also release pathogen-
associated molecular patterns (PAMPs) either inadver-
tently or as a consequence of plant degradative enzymes
(e.g. the release of chitin oligomers by plant chitinases).
Plants, in turn, appear to sense these PAMPs and damage
to their cell walls and activate a variety of defences,
including the production of reactive oxygen species
(ROS), the production and export of anti-microbial com-
pounds and fortification of their cell walls. In addition,
sensing PAMPs may activate intracellular defences like
the salicylic acid pathway, perhaps priming the plant for
the next stage of warfare.
In this commentary, we suggest that in addition to its
structural role, the plant cell wall also relays information
about the environment to the cell cytoplasm via signal
transduction pathway(s) that are patterned after PAMP
signallingpathways.Inthecaseofmicrobeattack,cellwall
fragments generated by either the plant or the microbe
activate plant defences, reinforcing the protection pro-
vided by plant cell wall. Experimental results supporting
thismodelarediscussedindetailbelow.Rolesfortheplant
cuticle in defence are not addressed in this article.
Pathogens use different invasion strategies
but similar weapons.
There are a variety of pathogen lifestyles from biotrophs
to necrotrophs, which influence the kinds of interactions
that occur at the plant cell wall. Necrotrophs release
copious amounts of cell wall degrading enzymes, pre-
sumably in an attempt to lyse plant cells before they can
mount an effective defence. Biotrophs, however, appear
to operate by stealth, minimizing damage to the host cell
wall. This difference in lifestyle is partially reflected in
the repertoire of cell wall degrading enzymes encoded by
the genomes of the two types of pathogens. The biotroph
Ustilago maydis encodes relatively few cell wall degrading
enzymes [5]; while a necrotroph like Erwinia spp., which
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causes soft rot diseases, produces a broad spectrum of
lipases and cutinases to degrade the host cuticle, pecti-
nases to increase accessibility for other enzymes like
cellulases and xylanases and several other hydrolases to
break down the hemicellulose chains [6]. Interestingly,
the nematode roundworm, Globodera rostochiensis, pro-
duces expansin proteins to loosen the cell wall [7].
Although this review is focused mainly on those patho-
gens that must damage the host cell wall to gain access to
the protoplast, similar observations have been made for a
number of bacterial pathogens that reside in the apoplast
and do not invade the plant cell protoplast (see Figure 1).
These bacterial pathogens release PAMPs into the cell
wall space that not only can be detected by specialized
PAMP receptors in the plant plasma membrane, the
pattern recognition receptors (PRR) (see article in this
issue by Zipfel) but also may be detected indirectly by
their impact on the plant cell wall. A number of bacterial
pathogens release cell wall degrading enzymes [6] and
electron micrographs show minor amounts of plant cell
wall degradation adjacent to populations of bacteria [8].
Plant surveillance of pathogen invasion:
elicitors inform about enemies
Plants possess different mechanisms to detect pathogen
invasion. They can directly detect pathogen presence by
non-self recognition of PAMPs or they can monitor the
integrity of their own cell wall (i.e. ‘intact self’ or
‘degraded self’). The cellular responses to PAMPs such
asflg22orchitinandcellwallperturbationareverysimilar
(e.g. induction of ROS, overproduction of callose and
lignin). Thus, PAMP signalling can be used to study
the less characterized cell wall integrity signalling. Below
we focus on what is known about cell wall integrity
signalling.
Cell wall integrity signalling is best characterised in yeast,
in which cell wall damage is sensed by the plasma
membrane-resident WSC1, WSC2, WSC3 and MID2
receptors[9].Preciselyhowthecellwalldamageissensed
by these receptors is unknown. The sensors recruit
Rom1/2 GEFs (guanyl-nucleotide exchange factors) to
the plasma membrane and then stimulate nucleotide
exchange on the Ras-like G-protein RHO1. Activation
of RHO1 directly regulates a b-1,3-glucan synthase
(Fks1), which synthesizes one of the major polysacchar-
ide components of the yeast cell wall. Activated RHO1
also initiates a MAP-kinase cascade that activates the
transcription factors RLM1 and the SBF complex (Swi4/
Swi6) to regulate the expression of genes required for cell
wall repair and cell cycle transitions. Actin cytoskeleton
rearrangements and post-translational activation of the
Fks1 glucan synthase are also triggered. Thus, cell wall
integrity in yeast is focused on sensing cell wall damage
and the activation of genes and enzymes needed to repair
the cell wall.
In cell wall integrity sensing in plants, neither the signal-
ling molecule nor the signal transduction event is known,
although there are hints that damage to polysaccharides
triggers responses in plants. No homologues of WSC1 or
MID2 receptors have been found; however, plant gen-
omes encode several potential receptor-like kinases
(RLK) that could act as cell wall integrity sensors. Cell
wall damage (e.g. in cell wall mutants or following treat-
ment with cell wall damaging agents) can be associated
with elevated levels of plant defence hormones like
Host–pathogen warfare at the plant cell wall He ´maty, Cherk and Somerville 407
Figure 1
Electron micrographs of different pathogens infecting plant cells. (a) Blumeria graminis f. sp. hordei fungal appressorium penetrating the cell wall of a
barley leaf epidermal cell. One can observe the degradation of the plant cell wall (CW) by the penetration peg and the host cellular responses involving
cytoplasm polarization and deposition of an electron dense cell wall apposition beneath the penetration site. Adapted from [59]. (b) A Pseudomonas
syringae pv phaseolicola bacterium (b) infecting in the intercellular space (IS) of lettuce cells. The arrow points to a H2O2deposition at the papilla plant
cell membrane interface. Adapted from [60]. Bar = 0.5 mm (c) Penetration of root cells by the Soybean Cyst Nematode Heterodera glycines. Image
courtesy of David Chitwood, Nematology Laboratory, USDA. isc: initial syncitial cell; St: stylet; FP: feeding plug; Bar = 1 mm. White star in (a) and (b)
represent the papilla deposited at the plant cell.
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salicylic acid (SA), ethylene (ET) or jasmonic acid (JA)
suggesting that one outcome of cell wall integrity signal-
ling is the activation of plant defences to limit pathogen
ingress at sites of pathogen attack and, pre-emptively, at
wound sites. An overview of what is known about sensing
each of the plant cell wall components is given below.
Sensing cell wall damage
We speculate that in plants, cell damage may be sensed
by one or more of the following: detection of damage to
polysaccharides, release of oligosaccharides, inhibition of
cell wall synthesis or assembly, or deformation of the
plasma membrane adjacent to damaged and weakened
cell walls.
As cellulose is the most abundant and strongest com-
ponent of the plant cell wall, pathogens target it for
degradation to facilitate penetration and to generate
glucose units as a food source. Mutations in CESAs
(encoding subunits of the cellulose synthase complex)
for either the primary or secondary cell wall induce a
number of stress and defence-like responses and enhance
resistance to certain pathogens. Cellulose deficiency in
the primary cell wall (e.g. in mutant cesa3 (=cev1)) elicits
ET and JA signalling and enhances resistance to bacteria,
fungi and aphids [10,11]. Similarly, inhibition of cellulose
synthesis with the herbicide isoxaben induces JA and SA
synthesis as well as some defence-associated genes [12??].
However, SA, ET and JA-independent resistance to the
soil-borne bacterium Ralstonia solanacearum and the
necrotrophic fungus
Plectosphaerella
observed in mutants deficient in cellulose in secondary
cell walls [13?]. By contrast, some Streptomyces species
release toxins (e.g. thaxtomin A) that inhibit cellulose
synthesis and facilitate virulence [14]. How the plant cell
detects a defect in cellulose synthesis is unknown; it may
monitorthe plasmamembrane-localized
synthase complex, the crystallinity or content of cellulose
produced or the generation of degraded cellulose frag-
ments [15??].
cucumerina
is
cellulose
Plant hemicelluloses include xyloglucans, xylans, glucur-
onoarabinoxylan, glucomannans and, in grasses, mixed-
linkage glucans [1]. Cellulose microfibrils are intercon-
nected by hemicelluloses to form the network that is
actively remodelled by plant enzymes (e.g. xyloglucan
endotransglycosylase) during cell expansion [16]. Inter-
estingly the mutant, resistant to Agrobacterium transform-
ation 4 (rat4), is deficient in the cellulose synthase-like A9
(CSLA9) [17]. CSLA proteins have mannan/glucomannan
synthase activity in vitro [18]. Additionally, mur3 mutants,
which are affected in a xyloglucan galactosyltransferase,
have increased SA levels in their petioles and are resistant
to Hyaloperonospora parasitica [19?].
Pectins are the most complex polysaccharides in plants,
composed of eleven different monosaccharides and
requiring a minimum of 67 enzymes for biosynthesis
[20]. The mutants, pmr5 (powdery mildew resistant5) and
pmr6, have changes in cell wall pectin composition
[21,22]. The penetration success of the powdery mildew
pathogen on these two mutants resembles wild type
suggesting that a change in cell wall digestibility by this
fungal pathogen per se was not responsible for the disease
resistance phenotype (Humphry and Somerville, unpub-
lished data). Thus, it is possible that the changed cell wall
or altered pectic fragments released during fungal attack
stimulate plant defences in these two mutants.
Cell wall elicitors
Pathogens secrete numerous glycosyl hydrolases and
lyases (e.g. b-1,4-and b-1,3-glucanases, pectinases, xylo-
sidases, arabinosidases and glucosidases with diverse
specificities) that degrade the plant cell wall and release
potential cell wall elicitors [2?]. For instance, degradation
of cellulose by b-1,4-glucanases generates cellodextrin
and degradation of the homogalacturonic domain of pec-
tins generates oligogalacturonic acid (OGA). Oligosac-
charides generated by cell wall degrading enzymes can
act as elicitors to trigger plant defences [23??]. OGA are
the best characterized plant cell wall derived elicitors.
Their ability to elicit a response depends on length
(degree of polymerization (dp) > 9) and degree of
methyl-esterification [24?]. Treatment of plants with
OGAcantrigger calcium influx,ROSproduction,changes
in gene expression and the ET and JA pathways [25,26].
In addition, plant genomes encode a wide range of plant
cell wall degrading enzymes, which probably play roles in
cell expansion but may also generate oligosaccharide
elicitors under stress conditions.
In addition to degradative enzymes, pathogens secrete
proteins with carbohydrate binding modules (CBM) but
lacking in enzymatic activity. The cellulose binding
elicitor lectin (CBEL) from Phytophthora parasitica nico-
tianae possesses two CBM1, which elicit a hypersensitive
response[27].Theroleoftheseproteinsisnotcompletely
understood. As has been proposed for the pectate lyase
domain of the bacterial HrpW effector, the CBM1
domains could serve to help anchor the pathogen to
the plant cell wall [28]. Alternately, the CBM1 domains
could disrupt associations between polysaccharides, such
as between glucan chains within cellulose microfibrils or
between cellulose and hemicelluloses. While the CBEL
protein may be a PAMP, it is also possible that the
induced defence responses result from disruption of
associations among glucan-containing polysaccharides
by the CBM. In some cases, the hydrolytic enzyme,
not its product, is recognized. A fungal ethylene-inducing
xylanase (EIX) protein is recognized by the tandem
tomato resistance genes, LeEix1 and LeEix2 [29]. Inter-
estingly, some pathogens appear to modify the host cell
wall to benefit infection. A secreted cellulose-binding
protein from the parasitic nematode Heterodera schachtii
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was shown to bind a host cell wall localized pectin
methylesterase(AtPME3)
mutants were more resistant to nematode penetration,
suggesting that interaction between the nematode
protein and host pectin methylesterase facilitates infec-
tions. Nematode infection also stimulates production of
plant cellulases and expansins, which are necessary for
cell wall relaxation during the formation of root syncytia
[31].
[30].Arabidopsis
pme3
Mechano-sensing of cell wall damage
The plant cell wall is physically linked to the plasma
membrane at discrete sites that form Hechtian strands
upon plasmolysis [32]. In mammalian cells, plasma mem-
brane-localized integrins bind to extracellular proteins
containing Arg-Gly-Asp (RGD) motifs, thus providing a
physical link between the plasma membrane and the
extracellular matrix [33]. Although no integrin-like
proteins have been found in plants, RGD-binding sites
at the plant plasma membrane have been detected [34].
These RGD-binding sites may link to the cell wall, as
treatment of plasmolyzed cells with RGD peptides dis-
rupts Hechtian strand formation [32]. Interestingly, treat-
ment with RGD peptides also decreases defence
responses during penetration and increases fungal
penetration success and intracellular growth [32]. Con-
sistent with this observation, pathogen effector proteins
with an RGD motif (e.g. the IPI-O protein from P.
infestans) have a similar effect on cell wall plasma mem-
brane connections [35]. Although there is no evidence
Host–pathogen warfare at the plant cell wall He ´maty, Cherk and Somerville409
Figure 2
Overview of the plant cell signalling components mediating responses upon pathogen attack. Plant cells possess a variety of membranes receptors
that perceive endogenous (e.g. plant cell wall (CW)-derived fragments = green stars) or exogenous (e.g. PAMP = pink stars) signals, which can be
either peptides or oligosaccharides (or both in the case of proteoglycans). A given ligand activates a specific receptor, which initiates downstream
signalling events. (1) Plant cells monitor their own cell wall through sensors like THE1. Inhibition of cellulose synthesis by the Streptomyces toxin
thaxtomin A or disruption of cellulose structure by a pathogen cellulose-binding protein could activate this pathway. (2) Additionally, host cells may
perceive pathogen intrusion through mechano-sensors. Stretching of the plasma membrane by invasive growth of a fungal haustorium could
potentially disrupt cell wall plasma membrane links via WAKs and other RLK, or act on stretch-activated Ca2+channels like Mca1. (3) Pathogen
invasion can also be detected by the presence of PAMPs (pathogen-derived peptides or cell wall fragments) and by plant derived cell wall fragments
generated by enzymes secreted by pathogens to aid penetration. Pathogens sometimes possess effectors (black stars) that can block defence
responses. The three types of sensors seem to converge on common signalling pathways. Responses involve rapid events such as post-protein
phosphorylation and activation (4), and slower events, like structural rearrangements of the cellular components (5) and induction of de novo protein
synthesis (6) to produce defensive compounds like phytoalexins, glucanases inhibitors and glucanases.
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supporting the idea that binding to RGD-containing
proteins (or other cell wall components) in the extracellu-
lar matrix by plasma membrane receptors transduces
changes in the mechanical properties of the plant cell
wall, this is an attractive idea.
Damageofthecellwallprobablyweakensthemechanical
strength of the wall and reduces the ability of the cell wall
to constrain turgor-driven expansion of the protoplast.
Mutantswith reduced cellulose levels commonlydevelop
swollen cells [12??]. This effect may be perceived as a
signal of pathogen invasion or wounding. Interestingly,
providing osmotic support to seedlings treated with a
cellulose synthase inhibitor reduced the cell swelling
phenotype and other cellular responses like lignin depo-
sition or the oxidative burst [12??]. Plasma membrane
protuberance upon cell wall perforation or invagination to
accommodate haustorium formation have both been
observed [36]. Such mechanical stress on the plasma
membrane could potentially activate mechano-sensing
ion channels (MscS-like channels [37]) or the Ca2+-per-
meable, stretch-activated channel mid1-complementing
activity 1 (Mca1), and then trigger changes in intracellular
Ca2+levels, initiating signalling events [38].
Plasma membrane-anchored receptors: the
embrasures of the fortress battlements
One can assume that perception of plant cell wall damage
involves plasma membrane-anchored proteins, like the
RLK (Figure 2). These RLKs may sense damage to the
plant cell wall directly via their extracellular domain or
may interact with a cell wall based receptor to form a
complex.
Wall-associated kinases (WAK) were the first cell wall
binding receptor kinases to be described [39]. WAK1
possesses an extracellular domain containing an epider-
mal growth factor (EGF)-like motif and appears to be
covalently bound to the cell wall [39]. The WAK1 protein
is released from the cell wall fraction by polygalacturo-
nase treatment, suggesting it is bound to a pectin ligand
[40]. The association between WAK1 and pectin is likely
to be covalent since it survives boiling in SDS and DTT
but the exact nature of the bond is unknown. Further-
more, WAK167–254aa, produced in yeast, binds to poly-
galacturonic acid (PGA) and OGA (dp > 9) in a Ca2+-
dependent manner [41].
Some WAK family members are involved in responses to
biotic stress [42]. The WAK1 transcript accumulates to
elevated levels upon pathogen infection or following
treatment with SA [42]. A more direct role in pathogen
resistance has been shown for WAKL22/RFO1 (RESIST-
ANCE TO FUSARIUM OXYSPORUM 1), which confers
broad spectrum resistance against Fusarium species [43].
Taken together, the evidence cited above suggests that
WAKLs are good candidates for receptors that monitor
pectin integrity during pathogen attack and trigger
defence responses. The predicted WAKL proteins are
highly similar in their cytoplasmic region but are more
divergent in their extracellular ligand-binding region,
suggesting that they could recognize different pectin
or, in some cases, glycine-rich protein ligands [44].
The
(CrRLK1-like) family members appear to perceive extra-
cellular signals and, in response, regulate various aspects
of development [45]. A member of this family, THE-
SEUS1 (THE1) from Arabidopsis, was identified in a
mutant screen for suppressors of the cellulose-deficient
mutant procuste1 (prc1 (=cesa6)). Mutation of THE1 par-
tially restores normal stature, and suppresses gene mis-
regulation and ectopic lignification in prc1 [15??]. THE1
is an attractive candidate for a cellulose integrity sensor,
although it is unknown whether THE1 acts directly by
binding to cellulose or indirectly by detecting the pleio-
tropic changes found in pcr1 mutants. No direct role in
pathogen resistance has been described for any CrRLK1L
family member. However, a few hints suggest these
RLKs might play such a role: (i) genes regulated by
THE1 in a prc1 background are stress-related, (ii) another
CrRLK1L member (At2g23200) has been shown, like the
PRRs FLS2 and EFR, to interact in vitro with the
bacterial effector AvrPto [46] and (iii) At5g39000 encodes
a protein with an RGD-binding motif [47].
Catharanthusroseus
receptor-likekinase1-like
Some RLKs have lectin-like extracellular domains [48].
Lectins appear to play a role in plant defence against
pathogens [49]. For instance, wheat-germ agglutinin
binds chitin and exhibits antifungal activity [50].
Additionally, lectin-containing RLK may bind polysac-
charides in the plant cell wall and, as proposed for WAK
and WAKL, sense changes in host cell walls upon
pathogen intrusion [47].
Cellular responses to changes in the cell wall
Signalling pathways
As noted above, cell wall damage is sensed by plants and
leads to the activation of various kinds of signalling
pathways. Deficiencies in cellulose, whether found in
mutants or in plants treated with cellulose synthesis
inhibitors, lead to the activation of the ET and JA
signalling pathways [10,11,12??], and in some cases, to
the activation of the SA or ABA pathways [12??,13?]. A
deficiency in wound and pathogen-induced callose depo-
sition is associated with hyper-activation of the SA signal-
ling pathway and resistance to some pathogens [51].
Similarly, SA levels were elevated in petioles with a
deficiency of xyloglucan galactosyltransferase [19?].
The release of OGA, like other PAMPs, leads to the
production of secondary signalling events, such as ROS
production, JA synthesis and changes in intracellular Ca2+
[25,26]. Changes in the resistance or susceptibility of
some plant cell wall mutants also suggest that other
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