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312
Early events in host–pathogen interactions
Murray Grant* and John Mansfield†
Research focused on early events in host–pathogen
interactions has provided new insights into fundamental
aspects of microbial pathogenicity and plant responses.
Considerable progress has been made in understanding
regulation of the delivery of pathogenicity determinants from
bacteria into plant cells, signal cascades involved in fungal
pathogenicity, the co-ordinating role of the plant cytoskeleton
in plant defence and calcium flux as a primary signalling
function during the hypersensitive reaction.
Addresses
Department of Biological Sciences, Wye College, Wye, Ashford,
Kent TN25 5AH, UK
*e-mail: m.grant@wye.ac.uk
†e-mail: j.mansfield@wye.ac.uk
Current Opinion in Plant Biology 1999, 2:312–319
http://biomednet.com/elecref/1369526600200312
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
avr
avirulence
[Ca2+]i cytosolic calcium concentration
HR
hypersensitive reaction
LRRP
leucine-rich repeat protein
MAPK mitogen-activated protein kinase
PKA
protein kinase A
R
resistance
ROS
reactive oxygen species
SLIK
signalling linker protein
vir
virulence
Introduction
The early events reviewed here are those occurring in
plants and also pathogens following inoculation. Scanning
recent research into plant diseases suggests that there has
perhaps been an unhealthy focus on resistance and in particular the hypersensitive reaction (HR) which is defined
as ‘the rapid death of plant cells at the infection site leading to restricted colonisation of the potential pathogen’.
There is no doubt that understanding the HR is an important target, but the key events in the establishment of
basic pathogenicity and susceptible interactions are comparatively poorly understood — particularly for obligate
fungal pathogens such as the rusts and mildews [1].
Varietal resistance involving gene-for-gene interactions is
typically expressed via the HR, but the more widespread
activation of non-host resistance does not usually involve
plant cell death — it involves highly co-ordinated localised
alterations to plant cell walls at challenge sites [1,2,3•].
Links are emerging, however, between avirulence (avr)
and virulence (vir) gene function with more and more
genes being found to have dual activity depending on the
presence or absence of the matching R gene in the plant
[4,5••]. The modes of action of potential virulence factors
remain to be determined, but an emerging hypothesis is
that vir gene function may be associated with suppression
of primitive mechanisms of resistance which do not
involve hypersensitive cell death [1].
Signal delivery by bacterial pathogens
Bacteria enter plants passively, often driven by rain-splash
through natural openings such as stomata. Exposure to the
micro-environment of the intercellular space in plant tissue induces expression of the type III secretion system,
which comprises components of the hrp gene cluster. The
hrp genes are required, both for pathogenicity and for the
ability to cause the HR in non-host and resistant host
plants [6]. The proteins encoded by hrp genes are homologous to those required for the delivery of virulence factors
by several mammalian pathogens, for example Yersinia [7].
No characteristic motifs have been identified in proteins
secreted by this route but recent experiments with Yersinia
show that the secretion signal may reside in untranslated
RNA upstream of the translation start [8,9••]. Operation of
such a co-ordinated translation and secretion system has
not yet been described in a plant pathogen [4,10].
Expression of hrp genes, and also coregulated avr genes, is
induced by growth in defined minimal media. But expression measured in the plant has regularly achieved levels far
greater than in vitro, implying some specific activation
beyond that achieved by low nutrient conditions [6]. In
Ralstonia, hrp regulation directly involves HrpB, which
shares homology with the AraC family of transcription activators [11]. Dissection of the regulatory pathway in
Ralstonia has also demonstrated the involvement of PrhA
(plant regulator of hrp genes), which controls interaction
with plants in an hrpB-independent manner [6,11,12••].
Although PrhA has significant similarity with siderophores,
which are typically associated with iron uptake, iron does
not appear to be the signal sensed in the plant.
Significantly, induction requires the presence of plant cells
(not just extracts) in suspension culture, implying a role for
bacterium/host cell contact.
Certain proteins with weak HR eliciting activity, such as
harpins from P. syringae and E. amylovora, and PopA from
R. solanacearum, are encoded by genes within or associated
with hrp clusters, and are readily secreted into culture
media. By contrast, there are many reports of failure to
detect similar secretion of other proteins, in particular
those encoded by avr genes, for example avrBs3 or avrB
from X. campestris pv. vesicatoria (Xcv) and P. syringae
respectively. The implication is that certain key virulence
determinants are not secreted into the intercellular space
but are directly transferred from bacteria into plant cells
using special components of the type III secretion system.
Two recent developments have, however, allowed secretion of Avr proteins to be demonstrated in vitro. The hrp
Early events in host–pathogen interactions Grant and Mansfield
cluster from E. chrysanthemi has proved to be ‘leaky’ and
has been expressed in E. coli, allowing secretion of AvrB to
be observed [13•]. More significantly, use of minimal
media with low pH and overexpression of regulatory hrp
genes, has allowed secretion of AvrB and also avrBs3 from
Xcv [10]. Having achieved secretion of known Avr proteins
it will now be possible to search for other proteins secreted by the same pathway, which may have virulence
functions and are, therefore, key players in the establishment of parasitism.
Secretion and delivery of Avr proteins, whether in vitro or in
the plant, seem to require the presence of a functional Hrp
pilus (Figure 1). Pili encoded by hrp genes have now been
identified in all major groups of plant pathogens. Purified
HrpA protein from P. syringae pv. tomato was found to
reassemble into filamentous structures [14]. Localization of
HrpA by immunocytochemistry using transmission electron
microscopy has revealed the presence of the pilus within
plant cell walls in contact with bacteria (SY He and I Brown,
personal communication). The pilus appears to be capable
of crossing the cell wall barrier. The question remains, however, whether proteins are delivered through the pilus or if
penetration of the pilus into the wall merely anchors bacteria and allows localised secretion into the plant cell wall and
from there, via diffusion across the polysaccharide matrix,
into contact with the plant cell membrane.
Identification of virulence factors
313
Figure 1
Immunogold labelling of hrp pili (arrowed) produced by Pseudomonas
syringae pv. tomato grown on microscope grids in hrp inducing
medium. Antiserum to HrpA differentiates the straight, narrow hrp pili
from curved, thicker flagella (bottom right); bar, 0.5 µm. (I Brown,
E Roine, unpublished data).
and may be considered a PAI, has been located on a 154 kb
plasmid in P. syringae pv. phaseolicola. Loss of the plasmid
results in loss of virulence to previously susceptible cultivars; cured strains elicit the HR rather than causing disease
(R Jackson personal communication).
Expression of avr genes in plants with the matching
R gene leads to the HR in several interactions [15,16]. The
Avr proteins delivered by the hrp-dependent secretion system are, therefore, the elicitors of hypersensitive cell
death. Intriguingly, expression of avr genes has also been
reported to cause effects in plants which lack the complementary R genes [17••,18]. Transient expression of avr
genes has been the most common approach to examine
effects in plant cells but a more tractable system is provided by stable expression from a highly regulated promoter.
McNellis et al. [17••] describe such an approach, using
avrRpt2 expression via a glucocorticoid-induced promoter
in Arabidopsis. The expression of avrRpt2 has clear effects
in the absence of the matching R gene (in this case RPS2).
Induction of patchy necrosis and inhibition of root growth
were both observed [17••]. Use of the induced promoter
provides an elegant model to examine effects of Avr proteins on plant cells.
Genes required for pathogenicity but not for the ability to
cause the HR have also been identified in E. amylovora,
P. syringae pv. tomato (Pst) and Xcv. In the former two
species, homology and functional similarity have been
shown for the hrp-linked pathogenicity genes: the disease
specific locus dspEF [21•] (or previously dspAB [22]) in
E. amylovora and avrE in Pst. Intriguingly, the avrE locus
was so named because it determined the ability of Pst to
elicit the HR in the non-host soybean. In Xcv mutations in
the hpaA gene (hrp associated) abolish pathogenicity but
retain, in part, the ability to deliver the avrBs3-mediated
HR in pepper [23•]. Like AvrBs3, the HpaA protein was
found to contain nuclear localization signals which are
important for the interaction with the plant.
A dual function for avr genes in virulence as well as HRinduction is often not apparent because Avr mutants are
not compromised in pathogenicity [4]. Virulence functions
may be masked by redundancy and it may be that it is only
by deletion of large regions of the bacterial genome, equivalent to the pathogenicity islands (PAIs) in animal
pathogens [19], that loss of virulence will be achieved. The
possibility that avr genes may be located on mobile regions
in P. syringae has been proposed [20]. A region containing
several avr genes which has multiple virulence functions
Amongst fungal pathogens, Cladosporium fulvum, the
cause of tomato leaf mould, is unique in its intercellular
biotrophic growth habit. Proteins secreted by C. fulvum
into the leaf apoplast are avirulence gene products such
as Avr5 and Avr9, and virulence factors ECP1 and ECP2.
Mutation of the Ecp genes leads to loss of virulence and
spore production [5••]. By means of an elegant potato
virus X-derived expression system, genotypes of tomato
were identified that activated the HR following exposure to ECP2, which, therefore, has dual function as a
314
Biotic interactions
Figure 2
Localisation of actin filaments by rhodamine-phalloidin labelling in
onion epidermis 12 h after inoculation with Botrytis allii. Note the
spore and germ-tube (arrowed) on the surface overlying the plant cell
wall. Polymerised actin forms a network of filaments focusing towards
the site of attempted penetration: bar, 100 µm (adapted from [3•]).
determinant of both pathogenicity and avirulence.
Laugé et al. [5••] raise the possibility that separate
domains on ECP2 might determine the avirulence and
virulence activities. Nevertheless, because ECP2 is
required for full pathogenicity, the matching recognition
gene designated Cf-ECP2 should be efficient and
durable in protecting tomato against leaf mould disease.
Fungal infection structures
Bacterial surface features, such as hrp pili, have been
found to be significant factors in pathogenicity, but the
interaction between pathogenic fungi and their hosts is
structurally a far more complex process. To be successful, the fungi have to complete a well defined series of
developmental steps including (at least) spore germination, appressorium formation and plant cell wall
penetration. Attempts to unravel key determinants of
fungal pathogenicity have targeted events involved in
production of a specific infection structure, for example
the appressorium [24] or haustorium [25], or applied the
‘black box’ approach of mutant hunts based on insertional mutagenesis using tagged sequences [26•]. Most
detailed information comes from experiments with the
rice–blast fungus Magnaporthe grisea.
Several environmental cues have been reported to favour
appressorium differentiation. In the bean rust fungus,
topographical stimuli, including ridges as low as 0.5 µm in
height have been implicated. The mechanisms underlying the rapid perception of surface topography and
co-ordinated response remain to be discovered [24]. In
M. grisea, appressoria form on any hard hydrophobic surface and their production is stimulated by the addition of
cutin monomers or cAMP. Signalling through a cAMP
dependent pathway is critical for appressorium morphogenesis. Mutations in genes that encode either a G protein
α subunit or adenylate cyclase block appressorium differentiation and also have other effects on fungal growth.
The adenylate cyclase operates upstream of alternative
protein kinase A (PKA) holoenzymes which are separately required for growth, appressorium production and plant
cell wall penetration. Genetical analysis has demonstrated
that the cAMP-dependent signal interacts with a mitogenactivated protein (MAP) kinase called Pmk1 to allow
development of a mature appressorium [27]. Mutation in
a second MAP kinase encoded by MPS1 does not impair
differentiation of the appressorial structure but compromises function, as indicated by failure to penetrate
through the underlying plant cell wall [28••].
Analysis of intracellular haustoria, which are arguably the
most important structures required for nutrient uptake by
the economically devastating pathogens the rusts and
mildews, remains constrained by inherent problems in dealing with the obligate parasites. Purification of haustoria has,
however, allowed the isolation of rust genes induced during
the early stage of haustorium formation [25,29].
Plant defence — local cell wall alterations
An interesting feature of MPS1 mutants of M. grisea is
that their failure to penetrate through plant cell walls is
associated with the activation of papilla deposition and
localized plant cell wall alteration. This mechanism of
defence does not involve the HR and is commonly
observed in the expression of resistance which does not
involve gene-for-gene interactions. Cell walls are
strengthened at sites of attempted penetration by the
incorporation and oxidative cross-linking of proteins and
various phenolic subunits including hydroxycinnamic
acid amides. In barley, coumaroylagmatine derivatives
were identified at reaction sites expressing mlo-based
resistance to Erysiphe graminis and in onion, feruloylmethoxytyramine (FMT) and related amides
accumulated in epidermis challenged by Botrytis allii
[3•,30]. Alterations to the onion cell wall are preceded by
earlier polarisation of the cytoskeleton to sites of attempted penetration (Figure 2). The formation and movement
of vesicles containing FMT now provides a useful model
for the examination of exocytosis in plant cells and the
overall co-ordination of such a highly targeted response.
In bean leaves, the rapid and highly co-ordinated increases
occurring in activities of callose synthase and peroxidase,
and deposition of some of their substrates in mesophyll
cell walls and papillae adjacent to hrp mutant bacteria have
now been defined by immunocytochemistry [2]. Localized
reactions were associated with a minor burst of H2O2, but
accumulation of the reactive oxygen species (ROS) was
much less than observed during the HR [31•,32], supporting
Early events in host–pathogen interactions Grant and Mansfield
the idea that the activation of cell death programmes
requires high threshold levels of ROS. Both peroxidase
and neutrophil-like NADPH oxidase activities have been
implicated as generators of ROS such as H2O2 [33•,34].
The HR in gene-for-gene interactions: calcium
and confusing kinases
Recent overviews [1,35,36] attempt to place the HR in the
context of other defence responses and emphasise the
remarkable variability in the timing and nature of biochemical events occurring in different plant–pathogen
interactions (despite them all being described as examples
of the HR!). Although the timing and sequence of events
following gene-for-gene recognition may be very different,
they ultimately lead via alternative routes to hypersensitive cell death. Given the variability apparent at the
structural, cellular level it is not surprising that a bewildering range of early biochemical responses and pathways has
been described. Three key components have been promoted: elevated cytosolic calcium, Ca2+ binding proteins
(calmodulin) and protein phosphorylation (kinases).
Calcium and calmodulin
Evidence for an elevated cytosolic calcium ([Ca2+]i) triggering the activation of defence mechanisms has been
derived primarily from treatment of cell suspensions with
microbes [37,38] or elicitors [39,40•,41,42]. Although convenient, studies in culture do not necessarily reflect the
continued presence and contribution of the pathogen to
the interaction. It is clearly preferable to monitor calcium
fluxes within whole tissue. An in planta study, involving
ratio imaging of microinjected epidermal strips from cowpea after challenge with the basidospores of Uromyces,
provides strong evidence for a role of Ca2+ in hypersensitive cell death. HR specific changes were observed before
the fungus penetrated the plant cell wall and were probably mediated by peptide elicitors diffusing from invading
hyphae [43••].
Measurement of changes in [Ca 2+] i during the
P. syringae/Arabidopsis: AvrRpm1/RPM1 interaction using
aequorin-generated bioluminescence, has demonstrated
the in planta generation of a biphasic intracellular calcium signature (M Grant, I Brown, J Mansfield,
unpublished data). Our data suggest that elevation of
[Ca2+]i occurs almost instantaneously after delivery of
AvrRpm1 and is a primary function of the resistance
gene product RPM1 which contains leucine rich repeats
(LRRs). Calcium appears to serve as a post-recognition
molecular switch, transducing the initial RPM1-mediated response stimuli to multiple downstream effectors. A
role for RPM1 in activation of calcium channels is supported by the demonstration that RPM1 is a peripheral
membrane associated protein [44••], which interacts with
a putative membrane protein in the two-hybrid system
(cited in [44••]). Furthermore, the proposed requirement
for localisation of AvrRpm1 or AvrB to the plant plasma
membrane for activity [44••], suggests that calcium
315
elevation may be mediated via the actions of a membrane-localised protein complex.
In contrast to the intracellular AvrRpm1/RPM1 recognition, the C. fulvum/tomato interaction is mediated
through resistance gene (Cf) proteins such as Cf5 and
Cf9, which also contain LRRs, but are predicted to be
predominantly extracellular [45]. Elicitors from race 5 or
race 9 isolates of C. fulvum cause a rapid increase in
[Ca2+]i within minutes of their addition to tomato cells
containing the cognate resistance genes, Cf5 or Cf9
[42,46••]. Patch clamping studies have identified a plasma membrane Ca2+ channel activated upon challenge
with elicitor from race 5. This hyperpolarisation-activated
Ca2+ channel appears to be modulated by a G-protein
dependent mechanism [42]. Interestingly, race 5 concomitantly inhibits a plasma membrane Ca2+-ATPase,
thus providing a possible feedback mechanism limiting
Ca2+ efflux back to the apoplast [47] (see [48••] for an
excellent review). Elevated [Ca2+]i may also play a direct
role in activation of NADPH oxidase through binding to
the unique EF hand motifs in the amino-terminal region
of the Arabidopsis gp91phox homologue [49]. Significantly,
no direct association of Cf5 or Cf9 with their avirulence
gene products has been demonstrated. Based upon the
necrosis-inducing activity of several Avr9 peptides and
their association with a high affinity binding site present
in both susceptible and resistant tomato genotypes, it has
been proposed that an Avr9-binding protein mediates an
interaction between Avr9 and Cf9 [50•].
Calmodulin, Ca2+ activated phosphatases and kinases
probably integrate the elevated calcium signal into the
resultant diverse array of signalling pathways. The exquisite specificity of calcium-based responses is illustrated by
the rapid and selective transcriptional activation of specific calmodulin isoforms in soybean cell cultures, and the
effects of expression of a hyperactive mutant calmodulin in
transgenic tobacco [51••,52].
Confusing kinases
Surprisingly, the kinases activated during early signalling
events in gene-for-gene interactions are also involved in
non-specific elicitation of defence responses as well as
other diverse abiotic stimuli such as wounding and
mechanical stress [46••,53•,54•,55,56]. Challenge of transgenic tobacco cell suspensions expressing Cf9 with
purified Avr9 elicitor resulted in the rapid activation of
tobacco SIP kinase [46••], originally identified as a
MAPK activated by salicylic acid [57•], and WIPK, a
wound inducible kinase also required for jasmonatebased wound signal transduction [55]. The same kinases
were activated during N–gene recognition of tobacco
mosaic virus, but with significantly different activation
kinetics [53•]. In gene-for-gene interactions, and after
wounding, activation of the MAPKs appears to require
phosphotyrosine phosphorylation [46••,53•,54•,57•].
Identification of the tyrosine kinase necessary for this
316
Biotic interactions
Figure 3
Cladosporium fulvum
Apoplast
(1)
AVR9
Bacterial type III secretion
apparatus
Cf9
(4) AvrRpm1
Peronospora parasitica
Ca2+
(3) AvrPphB
Ca2+
(2) AvrPto
9
R
AV
Prf
AVR9
binding
protein
(SLIK)
SLIK
(NDR1)
SLIK
(?)
Avr
?
?
PBS1/?
SLIK
(EDS1)
Pto
AvrPto
RPS5/RPM1
RPP5
SLIK
(EDS1)
to
AvrRpm1
hB
Pto
Av
r
Pp
rP
Av
Cytoplasm
Ca2+
PBS1
?
RPM
1
Prf
RPS5
Calmodulin, calcium dependent protein kinases
?
Ca2+
RPP5
RPP14
RPS4
RPP2
(5) AvrRPP5
Current Opinion in Plant Biology
Speculative model for early signalling events occurring during selected
gene-for-gene interactions, based upon current genetic and
biochemical evidence. Components which have been characterised
are named (e.g. PBS1 [63•]), other proteins anticipated to be involved
are designated with question marks. As no direct interaction has been
demonstrated between avirulence gene products (Avr) and LRRPs
(indicated by proteins with tails), we propose an integrative pathway
involving signal linker proteins (SLIKs) and SLIK complexes, which may
function as chaperones or signalling intermediates in coupling
avirulence gene function to LRRPs. For example, in the simplest
interaction, the Avr9-binding protein acts as a SLIK linking Avr9 to the
LRRP, Cf9 and formation of the complex triggers Ca2+ influx (dashed
arrow). Similarly, Pto may function as part of a SLIK complex to
transduce AvrPto recognition to Prf. In our model, gene products
demonstrated to modify signalling pathways (EDS1 and NDR1) are
positioned as SLIKs to integrate specifically a distinct subset of Avr
signals towards their matching respective LRRPs (products of R
genes such as RPP5). As elevation of cytosolic calcium is one of the
earliest detectable events in these interactions, it is used to represent
a common secondary messenger. The sets of interactions described,
each involving their Avr protein, SLIK, additional SLIK complex
component and LRRP respectively, are as follows: 1. Avr9, SLIK
binding protein, no additional component, Cf9; 2. AvrPto, unknown
SLIK, Pto, Prf; 3. AvrPphB, NDR1 (as a SLIK), PBS1, RPS5;
4. AvrRpm1, NDR1, unknown component, RPM1; 5. Predicted
AvrRPP protein, EDS1 (as a SLIK), no additional component, RPP
resistance gene protein [50•,60,62,63•].
modification, and understanding its regulation is an
important future objective.
between such diverse stimuli and at what stages are irreversible cell death programmes activated?
The post-transcriptional activation of SIPK and WIPK,
although dependent upon elevated cytosolic Ca2+, calmodulin and phosphorylation, were not necessary for ROS
generation [46••]. However, a Ca2+-dependent protein kinase
has been implicated in the assembly of a functional NADPHoxidase complex during avr5/Cf5 mediated defence
responses in tomato [58]. These data suggest a very early
bifurcation of signalling pathways downstream of the Ca2+
mediated calmodulin signalling. The intriguing questions
now are: how do multifunctional kinases discriminate
Conclusions and a generalised model for early
interactions in plant cells
Despite enormous effort, no LRR protein (LRRP) resistance gene product has been shown to interact directly
with an Avr product. Perhaps the LRRPs instead interpret
a subset of specific signals generated from other cellular
proteins, which we designate signalling linker proteins
(SLIKs). The presence of an elicitor (e.g. Avr protein), or
its activity, may compromise the normal configuration or
function of a SLIK or a SLIK complex, and this would be
Early events in host–pathogen interactions Grant and Mansfield
recognised specifically by an LRRP which would, therefore, indirectly match the Avr protein. Comparative studies
on R gene clusters have revealed that the sequences
encoding putative solvent-exposed residues in the LRRPs
are hypervariable, exhibiting elevated ratios of nonsynonymous to synonymous substitutions [59]. Such a structure
would allow the dynamic evolution of diverse arrays of ligand acceptor sites.
The proposed interactions between Avr, SLIK and LRR
proteins are summarised in Figure 3, in which it is postulated that certain genes known to be required for
resistance, such as EDS1 and NDR1, many encode SLIKs
which operate upstream of the LRRPs. The role of EDS1
and NDR1 as SLIKs would explain their specific effects
on groups of gene-for-gene interactions involving different
LRRPs [60,61•]. The Pto kinase protein, which is known
to bind directly to AvrPto, may interact with a SLIK complex which includes both AvrPto and Prf (an LRRP), a
hierarchy which is supported by results from gain of function experiments with Pto [62]. Recent genetic evidence
suggests that the PBS1 protein, which is required for
AvrPphB/ RPS5-mediated resistance in Arabidopsis [63•],
may function in a role analogous to Pto and interact with
NDR1 in the SLIK complex. Whatever the final phenotype, the earliest response modulated by an LRRP appears
to be elevated [Ca2+]i.
With such a signalling hierarchy, certain non-specific elicitors, such as oligo-uronides, might affect SLIKs which
interact with more than one LRRP and, therefore, activate a diverse range of plant responses, only some of
which may lead to the HR [36]. Returning to our initial
point about unravelling susceptibility, it is envisaged that
Avr proteins are able to act as virulence factors by blocking the ability of the SLIK to interact with LRRPs and,
thereby, effectively suppressing plant defence responses.
As outlined in Figure 3, without SLIK activity, basic parasitim is established.
Acknowledgements
We wish to acknowledge support from the BBSRC and EC grant BIOCT97-2244. Thanks are also due to colleagues who provided details of
recent results.
References and recommended reading
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317
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1:109-114.
17.
••
McNellis TW, Mudgett MB, Li K, Aoyama T, Horvath D, Chua NH,
Staskawicz BJ: Glucocorticoid-inducible expression of a bacterial
avirulence gene in transgenic Arabidopsis induces hypersensitive
cell death. Plant J 1998, 14:247-257.
The stable and controlled expression of AvrRpt2 protein is a very significant
step towards understanding the function of Avr proteins in plants, with or
without the matching R gene.
18. Stevens C, Bennett MA, Athanassopoulos E, Tsiamis G, Taylor JD,
Mansfield JW: Sequence variations in alleles of the avirulence
gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola
lead to loss of recognition of the AvrPphE protein within bean
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29:165-177.
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318
Biotic interactions
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21. Bogdanove AJ, Kim JF, Wei Z, Kolchinsky P, Charkowski AO, Conlin
•
AK, Collmer A, Beer SV: Homology and functional similarity of an
hrp-linked pathogenicity locus, dspER, or Erwinia amylovora and
the avirulence locus avrE of Pseudomonas syringae pathovar
tomato. Proc Natl Acad Sci USA 1998, 95:1325-1330.
Demonstration of dual avirulence and pathogenicity functions in the dsp
locus. Highlights the emerging theme of common pathogenic ancestors to
diverse genera of phytopathogens (see also [22]).
22. Gaudriault S, Malandrin L, Paulin JP, Barny MA: DspA, an essential
pathogenicity factor of Erwinia amylovora showing homology with
AvrE of Pseudomonas syringae, is secreted via the Hrp secretion
pathway in a DspB-dependent way. Mol Microbiol 1997,
26:1057-1069.
23. Huguet E, Hahn K, Wengelnik K, Bonas U: hpaA mutants of
•
Xanthomonas campestris pv. vesicatoria are affected in
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hypersensitive reaction. Mol Microbiol 1998, 29:1379-1390.
Potential pathogenicity gene has emerged from detailed molecular characterisation of the hrp cluster in Xcv.
24. Dean RA: Signal pathways and appressorium morphogenesis.
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25. Hahn M, Mendgen K: Characterization of in planta-induced rust
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26. Balhadère PV, Foster AJ, Talbot NJ: Identification of pathogenicity
•
mutants of the rice blast fungus Magnaporthe grisea by insertional
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Indicates the potential of and also limitations to insertional mutagenesis as a
means of unravelling fungal pathogenicity.
27.
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28. Xu U-R, Staiger CJ, Hamer JE: Inactivation of the mitogen-activated
•• protein kinase Mps1 from the rice blast fungus prevents
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responses. Proc Natl Acad Sci USA 1998, 95:12713-12718.
Genetical analysis of M. grisea translated into biochemical mechanisms controlling development of functional appressoria.
29. Hahn M, Neef U, Struck C, Göttfert M, Mendgen K: A putative amino
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31. Bestwick CS, Brown IR, Bennett MH, Mansfield JW: Localization of
•
hydrogen peroxide accumulation during the hypersensitive
reaction of lettuce cells to Pseudomonas syringae pv.
phaseolicola. Plant Cell 1997, 9:209-221.
Important demonstration of the accumulation of H2O2 during the HR and
localized responses (without plant cell death) to hrp mutant bacteria.
Conclusions on the role of peroxidase as a ROS generator in this system may
be flawed because of lack of specificity of inhibitors used, KCN and azide.
32. Bestwick CS, Brown IR, Mansfield JW: Localized changes in
peroxidase activity accompany hydrogen perodixe generation
during the development of a nonhost hypersensitive reaction in
lettuce. Plant Physiol 1998, 118:1067-1078.
35. Mansfield JW, Bennett MH, Bestwick CS, Woods-Tor AM:
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fungal and bacterial pathogens: variation from recognition to
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36. Somssich IR, Hahlbrock K: Pathogen defence in plants — a
paradigm of biological complexity. Trends Plant Sci 1998, 3:86-90.
37.
Atkinson MM, Midland SL, Sims JJ, Keen NT: Syringolide 1 triggers
Ca2+ influx, K+efflux, and extracellular alkalization in soybean
cells carrying the disease-resistance gene RPg4. Plant Physiol
1996, 112:297-302.
38. Levine A, Pennell RI, Alvarez ME, Palmer R, Lamb C: Calciummediated apoptosis in a plant hypersensitive disease resistance
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39. Zimmermann S, Nurnberger T, Frachisse J-M, Wirtz W, Guern J,
Hedrich R, Scheel D: Receptor-mediated activation of a plant Ca2+permeable ion channel involved in pathogen defense. Proc Natl
Acad Sci USA 1997, 94:2751-2755.
40. Piedras P, Hammond-Kosack KE, Harrison K, Jones JDG: Rapid,
•
Cf9- and Avr9-dependent production of active oxygen species in
tobacco suspension cultures. Mol Plant–Microbe Interact 1998,
11:1155-1166.
Use of a heterologous cell suspension system to analyse early events in
Cf-9 mediated signalling without potential interference from numerous
Cf-9 homologues
41. Jabs T, Tschope M, Colling C, Hahlbrock K, Scheel D: Elicitor
stimulated ion fluxes and O2– from the oxidative burst are
essential components in triggering defense gene activation and
phytoalexin synthesis in parsley. Proc Natl Acad Sci USA 1997,
94:4800-4805.
42. Gelli A, Higgins VJ, Blumwald E: Activation of plant plasma
membrane Ca2+-permeable channels by race-specific fungal
elicitors. Plant Physiol 1997, 113:269-279.
43. Xu HX, Heath MC: Role of calcium in signal transduction during
•• the hypersensitive response caused by basidiospore-derived
infection of the cowpea rust fungus. Plant Cell 1998, 10:585-597.
Ratio imaging of microinjected epidermal strips from cowpea after challenge
with basidospores of Uromyces phaseoli provides the first compelling in
planta evidence for a role of elevated Ca2+ in the HR.
44. Boyes DC, Nam J, Dangl JL: The Arabidopsis thaliana RPM1
•• disease resistance gene product is a peripheral plasma
membrane protein that is degraded coincident with the
hypersensitive response. Proc Natl Acad Sci USA 1998,
95:15849-15854.
The authors provide the first evidence for the localisation of a NBS-LRR
resistance gene product. In addition they demonstrate the possibility of regulation of RPM1 in a typical Danglesque feedback-loop model.
45. Dixon MS, Hatzixanthis K, Jones DA, Harrison K, Jones JDG: The
tomato Cf-5 disease resistance gene and six homologs show
pronounced allelic variation in leucine-rich repeat copy number.
Plant Cell 1998, 10:1915-1925.
46. Romeis T, Piedras P, Zhang S, Klessig DF, Hirt H, Jones JD: Rapid
•• Avr9- and Cf-9-dependent activation of MAP kinases in tobacco
cell cultures and leaves. Convergence of resistance gene, elicitor,
wound, and salicylate responses. Plant Cell 1999, 11:273-288.
Evidence for gene-for-gene specific activation of two MAP kinases which are
not unique to defense responses but also implicated in other stress
response pathways. Additionally these these kinases exhibit calcium dependency and they are not involved in pathways signalling the generation of
NADPH dependent ROS.
47.
Lam CHB, Xing T, Higgens VJ, Blumwald E: Effect of race-specific
elicitors of cladosporium fulvum on the tomato plasma membrane
Ca2+-ATPase. Physiol Mol Plant Path 1998, 52:309-321.
33. Martinez C, Montillet JL, Bresson E, Agnel JP, Dai GH, Daniel JF,
•
Geiger JP, Nicole M: Apoplastic peroxidase generates superoxide
anions in cells of cotton cotyledons undergoing the hypersensitive
reaction to Xanthomonas campestris pv. malvaraceum race 18. Mol
Plant–Microbe Interact 1998, 11:1038-1047.
Good evidence for the role of peroxidase in generating ROS in cotton. In this
interaction NADPH oxidase does not seem to be as important.
48. Blumwald E, Aharon GS, Lam BC-H: Early signal transduction
•• pathways in plant–pathogen interactions. Trends Plant Sci 1998,
3:342-346.
Excellent biochemical perspective of early signalling events in
plant–pathogen interactions.
34. Bolwell GP, Davies DR, Gerrish C, Auh CK, Murphy TM:
Comparative biochemistry of the oxidative burst produced by
rose and French bean cells reveals two distinct mechanisms.
Plant Physiol 1998, 116:1379-1385.
49. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: A
plant homolog of the neturophil NADPH oxidase gp91phox subunit
gene encodes a plasma membrane protein with Ca2+ binding
motifs. Plant Cell 1998, 10:255-266.
Early events in host–pathogen interactions Grant and Mansfield
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50. Kooman-Gersmann M, Vogelsang R, Vossen P, Henno W, van den
•
Hooven EM, Honee G, de Wit PJGM: Correlation between binding
affinity and necrosis-inducing activity of mutant AVR9 peptide
elicitors. Plant Physiol 1998, 117:609-618.
The authors continue to accumulate strong evidence for the possibility that
Avr9 binds to a receptor other than Cf9.
57. Zang S, Klessig DF: The tobacco wounding-activated MAP kinase
•
is encoded by SIPK. Proc Natl Acad Sci USA 1998, 95:7433-7438.
Together with [46••,53•,54•], this paper provides compelling evidence for
SIPK signalling in gene-for-gene, non-specific elicitation and wounding.
Their results beg the question, how are these multiple stimuli integrated and
discriminated through the MAPK?
51. Heo WD, Lee SH, Kim MC, Kim JC, Chung WS, Chun HJ, Lee KJ,
•• Park CY, Park HC, Choi JY, Cho MJ: Involvement of specific
calmodulin isoforms in salicylic acid-independent activation of
plant disease resistance responses. Proc Natl Acad Sci USA
1999, 96:766-771.
Elegant experiments with transgenic tobacco implicate a specific subset of
calmodulin isoforms in signalling pathways leading to plant disease resistance.
58. Xing T, Higgins VJ, Blumwald E: Race-specific elicitors of
Cladosporium fulvum promote translocation of cytosolic
components of NADPH oxidase to the plasma membrane of
tomato cells. Plant Cell 1997, 9:249-259.
52. Harding SA, Roberts DM: Incompatible pathogen infection results
in enhanced reactive oxygen and cell death responses in
transgenic tobacco expressing a hyperactive mutant calmodulin.
Planta 1998, 206:253-258.
53. Zhang S, Klessig DF: Resistance gene N-mediated de novo
•
synthesis and activation of a tobacco mitogen-activated protein
kinase by tobacco mosaic virus infection. Proc Natl Acad Sci USA
1998, 95:7433-7438.
See annotation for [57•].
54. Zhang S, Du H, Klessig DF: Activation of the tobacco SIP kinase by
•
both a cell wall-derived carbohydrate elicitor and purified
proteinaceous elicitins from Phytophthora spp. Plant Cell 1998,
10:435-450.
See annotation for [57•].
55. Seoa S, Sanoc H,Ohashia Y: Jasmonate-based wound signal
transduction requires activation of WIPK, a tobacco mitogenactivated protein kinase. Plant Cell 1999, 11:289-298.
56. Ligterink W, Kroj T, zurNieden U, Hirt H, Scheel D: Receptormediated activation of a MAP kinase in pathogen defense of
plants. Science 1997, 276:2054-2057.
59. Michelmore RW, Meyers BC: Clusters of resistance genes in plants
evolve by divergent selection and a birth-and-death process.
Genome Research 1998, 8:1113-1130.
60. Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, Parker JE:
Different requirements for EDS1 and NDR1 by disease resistance
genes define at least two R gene-mediated signaling pathways in
Arabidopsis. Proc Natl Acad Sci USA 1998, 95:10306-10311.
61. Falk A, Feys B, Frost L, Jones JDG, Daniels MJ, Parker JE: EDS1, an
•
essential component of R gene-mediated disease resistance in
Arabidopsis has homology to eukaryotic lipases. Proc Natl Acad
Sci USA 1999, 96:3292-3297.
The cloning of EDS1, integral to signalling through a defined sub-set of R
genes, provides a base for biochemical dissection of pathways to resistance.
62. Rathjen JP, Chang JH, Staskawicz BJ, Michelmore RW:
Constitutively active Pto induces a Prf-dependent hypersensitive
response in the absence of AvrPto. EMBO J 1999, 3232-3240.
63. Warren RF, Merritt MM, Holub E, Innes RW: Identification of three
•
putative signal transduction genes involved in R gene-specified
disease resistance in Arabidopsis. Genetics 1999, 152:401-412.
Genetic dissection of a specific gene-for-gene pathway leads to identification of a locus, pbs1, which may be involved in specific regulation of
AvrPphB/RPS5 interaction.
Early events in host–pathogen interactions
Murray Grant* and John Mansfield†
Research focused on early events in host–pathogen
interactions has provided new insights into fundamental
aspects of microbial pathogenicity and plant responses.
Considerable progress has been made in understanding
regulation of the delivery of pathogenicity determinants from
bacteria into plant cells, signal cascades involved in fungal
pathogenicity, the co-ordinating role of the plant cytoskeleton
in plant defence and calcium flux as a primary signalling
function during the hypersensitive reaction.
Addresses
Department of Biological Sciences, Wye College, Wye, Ashford,
Kent TN25 5AH, UK
*e-mail: m.grant@wye.ac.uk
†e-mail: j.mansfield@wye.ac.uk
Current Opinion in Plant Biology 1999, 2:312–319
http://biomednet.com/elecref/1369526600200312
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
avr
avirulence
[Ca2+]i cytosolic calcium concentration
HR
hypersensitive reaction
LRRP
leucine-rich repeat protein
MAPK mitogen-activated protein kinase
PKA
protein kinase A
R
resistance
ROS
reactive oxygen species
SLIK
signalling linker protein
vir
virulence
Introduction
The early events reviewed here are those occurring in
plants and also pathogens following inoculation. Scanning
recent research into plant diseases suggests that there has
perhaps been an unhealthy focus on resistance and in particular the hypersensitive reaction (HR) which is defined
as ‘the rapid death of plant cells at the infection site leading to restricted colonisation of the potential pathogen’.
There is no doubt that understanding the HR is an important target, but the key events in the establishment of
basic pathogenicity and susceptible interactions are comparatively poorly understood — particularly for obligate
fungal pathogens such as the rusts and mildews [1].
Varietal resistance involving gene-for-gene interactions is
typically expressed via the HR, but the more widespread
activation of non-host resistance does not usually involve
plant cell death — it involves highly co-ordinated localised
alterations to plant cell walls at challenge sites [1,2,3•].
Links are emerging, however, between avirulence (avr)
and virulence (vir) gene function with more and more
genes being found to have dual activity depending on the
presence or absence of the matching R gene in the plant
[4,5••]. The modes of action of potential virulence factors
remain to be determined, but an emerging hypothesis is
that vir gene function may be associated with suppression
of primitive mechanisms of resistance which do not
involve hypersensitive cell death [1].
Signal delivery by bacterial pathogens
Bacteria enter plants passively, often driven by rain-splash
through natural openings such as stomata. Exposure to the
micro-environment of the intercellular space in plant tissue induces expression of the type III secretion system,
which comprises components of the hrp gene cluster. The
hrp genes are required, both for pathogenicity and for the
ability to cause the HR in non-host and resistant host
plants [6]. The proteins encoded by hrp genes are homologous to those required for the delivery of virulence factors
by several mammalian pathogens, for example Yersinia [7].
No characteristic motifs have been identified in proteins
secreted by this route but recent experiments with Yersinia
show that the secretion signal may reside in untranslated
RNA upstream of the translation start [8,9••]. Operation of
such a co-ordinated translation and secretion system has
not yet been described in a plant pathogen [4,10].
Expression of hrp genes, and also coregulated avr genes, is
induced by growth in defined minimal media. But expression measured in the plant has regularly achieved levels far
greater than in vitro, implying some specific activation
beyond that achieved by low nutrient conditions [6]. In
Ralstonia, hrp regulation directly involves HrpB, which
shares homology with the AraC family of transcription activators [11]. Dissection of the regulatory pathway in
Ralstonia has also demonstrated the involvement of PrhA
(plant regulator of hrp genes), which controls interaction
with plants in an hrpB-independent manner [6,11,12••].
Although PrhA has significant similarity with siderophores,
which are typically associated with iron uptake, iron does
not appear to be the signal sensed in the plant.
Significantly, induction requires the presence of plant cells
(not just extracts) in suspension culture, implying a role for
bacterium/host cell contact.
Certain proteins with weak HR eliciting activity, such as
harpins from P. syringae and E. amylovora, and PopA from
R. solanacearum, are encoded by genes within or associated
with hrp clusters, and are readily secreted into culture
media. By contrast, there are many reports of failure to
detect similar secretion of other proteins, in particular
those encoded by avr genes, for example avrBs3 or avrB
from X. campestris pv. vesicatoria (Xcv) and P. syringae
respectively. The implication is that certain key virulence
determinants are not secreted into the intercellular space
but are directly transferred from bacteria into plant cells
using special components of the type III secretion system.
Two recent developments have, however, allowed secretion of Avr proteins to be demonstrated in vitro. The hrp
Early events in host–pathogen interactions Grant and Mansfield
cluster from E. chrysanthemi has proved to be ‘leaky’ and
has been expressed in E. coli, allowing secretion of AvrB to
be observed [13•]. More significantly, use of minimal
media with low pH and overexpression of regulatory hrp
genes, has allowed secretion of AvrB and also avrBs3 from
Xcv [10]. Having achieved secretion of known Avr proteins
it will now be possible to search for other proteins secreted by the same pathway, which may have virulence
functions and are, therefore, key players in the establishment of parasitism.
Secretion and delivery of Avr proteins, whether in vitro or in
the plant, seem to require the presence of a functional Hrp
pilus (Figure 1). Pili encoded by hrp genes have now been
identified in all major groups of plant pathogens. Purified
HrpA protein from P. syringae pv. tomato was found to
reassemble into filamentous structures [14]. Localization of
HrpA by immunocytochemistry using transmission electron
microscopy has revealed the presence of the pilus within
plant cell walls in contact with bacteria (SY He and I Brown,
personal communication). The pilus appears to be capable
of crossing the cell wall barrier. The question remains, however, whether proteins are delivered through the pilus or if
penetration of the pilus into the wall merely anchors bacteria and allows localised secretion into the plant cell wall and
from there, via diffusion across the polysaccharide matrix,
into contact with the plant cell membrane.
Identification of virulence factors
313
Figure 1
Immunogold labelling of hrp pili (arrowed) produced by Pseudomonas
syringae pv. tomato grown on microscope grids in hrp inducing
medium. Antiserum to HrpA differentiates the straight, narrow hrp pili
from curved, thicker flagella (bottom right); bar, 0.5 µm. (I Brown,
E Roine, unpublished data).
and may be considered a PAI, has been located on a 154 kb
plasmid in P. syringae pv. phaseolicola. Loss of the plasmid
results in loss of virulence to previously susceptible cultivars; cured strains elicit the HR rather than causing disease
(R Jackson personal communication).
Expression of avr genes in plants with the matching
R gene leads to the HR in several interactions [15,16]. The
Avr proteins delivered by the hrp-dependent secretion system are, therefore, the elicitors of hypersensitive cell
death. Intriguingly, expression of avr genes has also been
reported to cause effects in plants which lack the complementary R genes [17••,18]. Transient expression of avr
genes has been the most common approach to examine
effects in plant cells but a more tractable system is provided by stable expression from a highly regulated promoter.
McNellis et al. [17••] describe such an approach, using
avrRpt2 expression via a glucocorticoid-induced promoter
in Arabidopsis. The expression of avrRpt2 has clear effects
in the absence of the matching R gene (in this case RPS2).
Induction of patchy necrosis and inhibition of root growth
were both observed [17••]. Use of the induced promoter
provides an elegant model to examine effects of Avr proteins on plant cells.
Genes required for pathogenicity but not for the ability to
cause the HR have also been identified in E. amylovora,
P. syringae pv. tomato (Pst) and Xcv. In the former two
species, homology and functional similarity have been
shown for the hrp-linked pathogenicity genes: the disease
specific locus dspEF [21•] (or previously dspAB [22]) in
E. amylovora and avrE in Pst. Intriguingly, the avrE locus
was so named because it determined the ability of Pst to
elicit the HR in the non-host soybean. In Xcv mutations in
the hpaA gene (hrp associated) abolish pathogenicity but
retain, in part, the ability to deliver the avrBs3-mediated
HR in pepper [23•]. Like AvrBs3, the HpaA protein was
found to contain nuclear localization signals which are
important for the interaction with the plant.
A dual function for avr genes in virulence as well as HRinduction is often not apparent because Avr mutants are
not compromised in pathogenicity [4]. Virulence functions
may be masked by redundancy and it may be that it is only
by deletion of large regions of the bacterial genome, equivalent to the pathogenicity islands (PAIs) in animal
pathogens [19], that loss of virulence will be achieved. The
possibility that avr genes may be located on mobile regions
in P. syringae has been proposed [20]. A region containing
several avr genes which has multiple virulence functions
Amongst fungal pathogens, Cladosporium fulvum, the
cause of tomato leaf mould, is unique in its intercellular
biotrophic growth habit. Proteins secreted by C. fulvum
into the leaf apoplast are avirulence gene products such
as Avr5 and Avr9, and virulence factors ECP1 and ECP2.
Mutation of the Ecp genes leads to loss of virulence and
spore production [5••]. By means of an elegant potato
virus X-derived expression system, genotypes of tomato
were identified that activated the HR following exposure to ECP2, which, therefore, has dual function as a
314
Biotic interactions
Figure 2
Localisation of actin filaments by rhodamine-phalloidin labelling in
onion epidermis 12 h after inoculation with Botrytis allii. Note the
spore and germ-tube (arrowed) on the surface overlying the plant cell
wall. Polymerised actin forms a network of filaments focusing towards
the site of attempted penetration: bar, 100 µm (adapted from [3•]).
determinant of both pathogenicity and avirulence.
Laugé et al. [5••] raise the possibility that separate
domains on ECP2 might determine the avirulence and
virulence activities. Nevertheless, because ECP2 is
required for full pathogenicity, the matching recognition
gene designated Cf-ECP2 should be efficient and
durable in protecting tomato against leaf mould disease.
Fungal infection structures
Bacterial surface features, such as hrp pili, have been
found to be significant factors in pathogenicity, but the
interaction between pathogenic fungi and their hosts is
structurally a far more complex process. To be successful, the fungi have to complete a well defined series of
developmental steps including (at least) spore germination, appressorium formation and plant cell wall
penetration. Attempts to unravel key determinants of
fungal pathogenicity have targeted events involved in
production of a specific infection structure, for example
the appressorium [24] or haustorium [25], or applied the
‘black box’ approach of mutant hunts based on insertional mutagenesis using tagged sequences [26•]. Most
detailed information comes from experiments with the
rice–blast fungus Magnaporthe grisea.
Several environmental cues have been reported to favour
appressorium differentiation. In the bean rust fungus,
topographical stimuli, including ridges as low as 0.5 µm in
height have been implicated. The mechanisms underlying the rapid perception of surface topography and
co-ordinated response remain to be discovered [24]. In
M. grisea, appressoria form on any hard hydrophobic surface and their production is stimulated by the addition of
cutin monomers or cAMP. Signalling through a cAMP
dependent pathway is critical for appressorium morphogenesis. Mutations in genes that encode either a G protein
α subunit or adenylate cyclase block appressorium differentiation and also have other effects on fungal growth.
The adenylate cyclase operates upstream of alternative
protein kinase A (PKA) holoenzymes which are separately required for growth, appressorium production and plant
cell wall penetration. Genetical analysis has demonstrated
that the cAMP-dependent signal interacts with a mitogenactivated protein (MAP) kinase called Pmk1 to allow
development of a mature appressorium [27]. Mutation in
a second MAP kinase encoded by MPS1 does not impair
differentiation of the appressorial structure but compromises function, as indicated by failure to penetrate
through the underlying plant cell wall [28••].
Analysis of intracellular haustoria, which are arguably the
most important structures required for nutrient uptake by
the economically devastating pathogens the rusts and
mildews, remains constrained by inherent problems in dealing with the obligate parasites. Purification of haustoria has,
however, allowed the isolation of rust genes induced during
the early stage of haustorium formation [25,29].
Plant defence — local cell wall alterations
An interesting feature of MPS1 mutants of M. grisea is
that their failure to penetrate through plant cell walls is
associated with the activation of papilla deposition and
localized plant cell wall alteration. This mechanism of
defence does not involve the HR and is commonly
observed in the expression of resistance which does not
involve gene-for-gene interactions. Cell walls are
strengthened at sites of attempted penetration by the
incorporation and oxidative cross-linking of proteins and
various phenolic subunits including hydroxycinnamic
acid amides. In barley, coumaroylagmatine derivatives
were identified at reaction sites expressing mlo-based
resistance to Erysiphe graminis and in onion, feruloylmethoxytyramine (FMT) and related amides
accumulated in epidermis challenged by Botrytis allii
[3•,30]. Alterations to the onion cell wall are preceded by
earlier polarisation of the cytoskeleton to sites of attempted penetration (Figure 2). The formation and movement
of vesicles containing FMT now provides a useful model
for the examination of exocytosis in plant cells and the
overall co-ordination of such a highly targeted response.
In bean leaves, the rapid and highly co-ordinated increases
occurring in activities of callose synthase and peroxidase,
and deposition of some of their substrates in mesophyll
cell walls and papillae adjacent to hrp mutant bacteria have
now been defined by immunocytochemistry [2]. Localized
reactions were associated with a minor burst of H2O2, but
accumulation of the reactive oxygen species (ROS) was
much less than observed during the HR [31•,32], supporting
Early events in host–pathogen interactions Grant and Mansfield
the idea that the activation of cell death programmes
requires high threshold levels of ROS. Both peroxidase
and neutrophil-like NADPH oxidase activities have been
implicated as generators of ROS such as H2O2 [33•,34].
The HR in gene-for-gene interactions: calcium
and confusing kinases
Recent overviews [1,35,36] attempt to place the HR in the
context of other defence responses and emphasise the
remarkable variability in the timing and nature of biochemical events occurring in different plant–pathogen
interactions (despite them all being described as examples
of the HR!). Although the timing and sequence of events
following gene-for-gene recognition may be very different,
they ultimately lead via alternative routes to hypersensitive cell death. Given the variability apparent at the
structural, cellular level it is not surprising that a bewildering range of early biochemical responses and pathways has
been described. Three key components have been promoted: elevated cytosolic calcium, Ca2+ binding proteins
(calmodulin) and protein phosphorylation (kinases).
Calcium and calmodulin
Evidence for an elevated cytosolic calcium ([Ca2+]i) triggering the activation of defence mechanisms has been
derived primarily from treatment of cell suspensions with
microbes [37,38] or elicitors [39,40•,41,42]. Although convenient, studies in culture do not necessarily reflect the
continued presence and contribution of the pathogen to
the interaction. It is clearly preferable to monitor calcium
fluxes within whole tissue. An in planta study, involving
ratio imaging of microinjected epidermal strips from cowpea after challenge with the basidospores of Uromyces,
provides strong evidence for a role of Ca2+ in hypersensitive cell death. HR specific changes were observed before
the fungus penetrated the plant cell wall and were probably mediated by peptide elicitors diffusing from invading
hyphae [43••].
Measurement of changes in [Ca 2+] i during the
P. syringae/Arabidopsis: AvrRpm1/RPM1 interaction using
aequorin-generated bioluminescence, has demonstrated
the in planta generation of a biphasic intracellular calcium signature (M Grant, I Brown, J Mansfield,
unpublished data). Our data suggest that elevation of
[Ca2+]i occurs almost instantaneously after delivery of
AvrRpm1 and is a primary function of the resistance
gene product RPM1 which contains leucine rich repeats
(LRRs). Calcium appears to serve as a post-recognition
molecular switch, transducing the initial RPM1-mediated response stimuli to multiple downstream effectors. A
role for RPM1 in activation of calcium channels is supported by the demonstration that RPM1 is a peripheral
membrane associated protein [44••], which interacts with
a putative membrane protein in the two-hybrid system
(cited in [44••]). Furthermore, the proposed requirement
for localisation of AvrRpm1 or AvrB to the plant plasma
membrane for activity [44••], suggests that calcium
315
elevation may be mediated via the actions of a membrane-localised protein complex.
In contrast to the intracellular AvrRpm1/RPM1 recognition, the C. fulvum/tomato interaction is mediated
through resistance gene (Cf) proteins such as Cf5 and
Cf9, which also contain LRRs, but are predicted to be
predominantly extracellular [45]. Elicitors from race 5 or
race 9 isolates of C. fulvum cause a rapid increase in
[Ca2+]i within minutes of their addition to tomato cells
containing the cognate resistance genes, Cf5 or Cf9
[42,46••]. Patch clamping studies have identified a plasma membrane Ca2+ channel activated upon challenge
with elicitor from race 5. This hyperpolarisation-activated
Ca2+ channel appears to be modulated by a G-protein
dependent mechanism [42]. Interestingly, race 5 concomitantly inhibits a plasma membrane Ca2+-ATPase,
thus providing a possible feedback mechanism limiting
Ca2+ efflux back to the apoplast [47] (see [48••] for an
excellent review). Elevated [Ca2+]i may also play a direct
role in activation of NADPH oxidase through binding to
the unique EF hand motifs in the amino-terminal region
of the Arabidopsis gp91phox homologue [49]. Significantly,
no direct association of Cf5 or Cf9 with their avirulence
gene products has been demonstrated. Based upon the
necrosis-inducing activity of several Avr9 peptides and
their association with a high affinity binding site present
in both susceptible and resistant tomato genotypes, it has
been proposed that an Avr9-binding protein mediates an
interaction between Avr9 and Cf9 [50•].
Calmodulin, Ca2+ activated phosphatases and kinases
probably integrate the elevated calcium signal into the
resultant diverse array of signalling pathways. The exquisite specificity of calcium-based responses is illustrated by
the rapid and selective transcriptional activation of specific calmodulin isoforms in soybean cell cultures, and the
effects of expression of a hyperactive mutant calmodulin in
transgenic tobacco [51••,52].
Confusing kinases
Surprisingly, the kinases activated during early signalling
events in gene-for-gene interactions are also involved in
non-specific elicitation of defence responses as well as
other diverse abiotic stimuli such as wounding and
mechanical stress [46••,53•,54•,55,56]. Challenge of transgenic tobacco cell suspensions expressing Cf9 with
purified Avr9 elicitor resulted in the rapid activation of
tobacco SIP kinase [46••], originally identified as a
MAPK activated by salicylic acid [57•], and WIPK, a
wound inducible kinase also required for jasmonatebased wound signal transduction [55]. The same kinases
were activated during N–gene recognition of tobacco
mosaic virus, but with significantly different activation
kinetics [53•]. In gene-for-gene interactions, and after
wounding, activation of the MAPKs appears to require
phosphotyrosine phosphorylation [46••,53•,54•,57•].
Identification of the tyrosine kinase necessary for this
316
Biotic interactions
Figure 3
Cladosporium fulvum
Apoplast
(1)
AVR9
Bacterial type III secretion
apparatus
Cf9
(4) AvrRpm1
Peronospora parasitica
Ca2+
(3) AvrPphB
Ca2+
(2) AvrPto
9
R
AV
Prf
AVR9
binding
protein
(SLIK)
SLIK
(NDR1)
SLIK
(?)
Avr
?
?
PBS1/?
SLIK
(EDS1)
Pto
AvrPto
RPS5/RPM1
RPP5
SLIK
(EDS1)
to
AvrRpm1
hB
Pto
Av
r
Pp
rP
Av
Cytoplasm
Ca2+
PBS1
?
RPM
1
Prf
RPS5
Calmodulin, calcium dependent protein kinases
?
Ca2+
RPP5
RPP14
RPS4
RPP2
(5) AvrRPP5
Current Opinion in Plant Biology
Speculative model for early signalling events occurring during selected
gene-for-gene interactions, based upon current genetic and
biochemical evidence. Components which have been characterised
are named (e.g. PBS1 [63•]), other proteins anticipated to be involved
are designated with question marks. As no direct interaction has been
demonstrated between avirulence gene products (Avr) and LRRPs
(indicated by proteins with tails), we propose an integrative pathway
involving signal linker proteins (SLIKs) and SLIK complexes, which may
function as chaperones or signalling intermediates in coupling
avirulence gene function to LRRPs. For example, in the simplest
interaction, the Avr9-binding protein acts as a SLIK linking Avr9 to the
LRRP, Cf9 and formation of the complex triggers Ca2+ influx (dashed
arrow). Similarly, Pto may function as part of a SLIK complex to
transduce AvrPto recognition to Prf. In our model, gene products
demonstrated to modify signalling pathways (EDS1 and NDR1) are
positioned as SLIKs to integrate specifically a distinct subset of Avr
signals towards their matching respective LRRPs (products of R
genes such as RPP5). As elevation of cytosolic calcium is one of the
earliest detectable events in these interactions, it is used to represent
a common secondary messenger. The sets of interactions described,
each involving their Avr protein, SLIK, additional SLIK complex
component and LRRP respectively, are as follows: 1. Avr9, SLIK
binding protein, no additional component, Cf9; 2. AvrPto, unknown
SLIK, Pto, Prf; 3. AvrPphB, NDR1 (as a SLIK), PBS1, RPS5;
4. AvrRpm1, NDR1, unknown component, RPM1; 5. Predicted
AvrRPP protein, EDS1 (as a SLIK), no additional component, RPP
resistance gene protein [50•,60,62,63•].
modification, and understanding its regulation is an
important future objective.
between such diverse stimuli and at what stages are irreversible cell death programmes activated?
The post-transcriptional activation of SIPK and WIPK,
although dependent upon elevated cytosolic Ca2+, calmodulin and phosphorylation, were not necessary for ROS
generation [46••]. However, a Ca2+-dependent protein kinase
has been implicated in the assembly of a functional NADPHoxidase complex during avr5/Cf5 mediated defence
responses in tomato [58]. These data suggest a very early
bifurcation of signalling pathways downstream of the Ca2+
mediated calmodulin signalling. The intriguing questions
now are: how do multifunctional kinases discriminate
Conclusions and a generalised model for early
interactions in plant cells
Despite enormous effort, no LRR protein (LRRP) resistance gene product has been shown to interact directly
with an Avr product. Perhaps the LRRPs instead interpret
a subset of specific signals generated from other cellular
proteins, which we designate signalling linker proteins
(SLIKs). The presence of an elicitor (e.g. Avr protein), or
its activity, may compromise the normal configuration or
function of a SLIK or a SLIK complex, and this would be
Early events in host–pathogen interactions Grant and Mansfield
recognised specifically by an LRRP which would, therefore, indirectly match the Avr protein. Comparative studies
on R gene clusters have revealed that the sequences
encoding putative solvent-exposed residues in the LRRPs
are hypervariable, exhibiting elevated ratios of nonsynonymous to synonymous substitutions [59]. Such a structure
would allow the dynamic evolution of diverse arrays of ligand acceptor sites.
The proposed interactions between Avr, SLIK and LRR
proteins are summarised in Figure 3, in which it is postulated that certain genes known to be required for
resistance, such as EDS1 and NDR1, many encode SLIKs
which operate upstream of the LRRPs. The role of EDS1
and NDR1 as SLIKs would explain their specific effects
on groups of gene-for-gene interactions involving different
LRRPs [60,61•]. The Pto kinase protein, which is known
to bind directly to AvrPto, may interact with a SLIK complex which includes both AvrPto and Prf (an LRRP), a
hierarchy which is supported by results from gain of function experiments with Pto [62]. Recent genetic evidence
suggests that the PBS1 protein, which is required for
AvrPphB/ RPS5-mediated resistance in Arabidopsis [63•],
may function in a role analogous to Pto and interact with
NDR1 in the SLIK complex. Whatever the final phenotype, the earliest response modulated by an LRRP appears
to be elevated [Ca2+]i.
With such a signalling hierarchy, certain non-specific elicitors, such as oligo-uronides, might affect SLIKs which
interact with more than one LRRP and, therefore, activate a diverse range of plant responses, only some of
which may lead to the HR [36]. Returning to our initial
point about unravelling susceptibility, it is envisaged that
Avr proteins are able to act as virulence factors by blocking the ability of the SLIK to interact with LRRPs and,
thereby, effectively suppressing plant defence responses.
As outlined in Figure 3, without SLIK activity, basic parasitim is established.
Acknowledgements
We wish to acknowledge support from the BBSRC and EC grant BIOCT97-2244. Thanks are also due to colleagues who provided details of
recent results.
References and recommended reading
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•
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