Protein kinases in the plant defence response Tina Romeis
Protein kinases play a central role in signalling during pathogen
plasma membrane are responsible for signal perception.
recognition and the subsequent activation of plant defence
Upon pathogen recognition, signalling events become
mechanisms. Members of different kinase subfamilies, such as
initiated that trigger early cellular responses such as
calcium-dependent protein kinases and MAP kinases, are
changes in ion fluxes, synthesis of reactive oxygen species
involved. Nevertheless, often, only a single component of a
and changes in gene transcription, and often a localised
signalling cascade in an experimental plant system has been
hypersensitive cell death is observed as necrotic lesions
characterised. The future challenge is to understand how these
(Figure 1). Delayed defence responses include the pro-
kinases work, which cellular responses they mediate, and how
duction of antimicrobial compounds, cell wall fortification,
they fit into the bigger picture of defence signalling. This
and the activation of systemic acquired resistance (SAR),
challenge has become increasingly feasible with the recent
which reflects a long-lasting resistance that is established
introduction of new techniques: these techniques include
in non-infected areas of the plant [1,2].
reverse genetics, which will allow the allocation of biologicalfunction to kinase isoforms, (phospho) proteomics combined
Over the past few years, protein kinases have been identi-
with mass spectrometry, and transient expression of kinases in
fied after nonrace- and race-specific elicitation for each step
a (constitutively) active form, mimicking the induction of
in the induction of defence responses: they participate in
defence responses in a biological system.
the direct perception of elicitors and Avr products, theymediate signalling required for the induction of defence
mechanisms, including the activation of transcription fac-
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich,
tors and systemic responses, and they function as negative
NR4 7UH, UK; e-mail: email@example.com and
regulators or are involved in desensitisation of defence
Max-Planck-Institüt fur Züchtungsforschung, Abt. Molekulare
responses (Figure 2). A future objective is to identify the
Phytopathologie, Carl-von-Linne-weg 10, 50829 Köln, Germany;e-mail: firstname.lastname@example.org
connections between single signalling components func-tioning, for example, in direct perception of pathogens with
Current Opinion in Plant Biology 2001, 4:407–414
those involved in downstream signal transduction. This
requires the exploration of missing links upstream, down-
2001 Elsevier Science Ltd. All rights reserved.
stream and sideways of a given protein kinase with the aimof characterising regulators, phosphorylation targets, and
interacting partners that may sequester the enzyme into
specific phosphorylation cascades or signalling complexes.
This also requires a greater emphasis on biochemical
analysis and reverse genetics than has occurred in the past.
In this review, I concentrate on the most recent develop-
ments in which protein kinases have been associated
directly with plant resistance and the activation of
defence-related responses. In particular, I focus on techni-
cal aspects that may become useful for future research by
addressing key issues of protein kinase signalling in vivowithin a biological system. For more detailed information
on the characterisation of specific protein kinases in a
The plant’s surveillance system for pathogen attack is
given plant–pathogen interaction, I direct the reader to
based on early recognition of the invading organism and
recently published reviews [3,4•].
the activation of defence mechanisms that result in arrestof further invasion and resistance of the plant. Recognition
Protein kinases involved in the recognition of
is accomplished by the detection of elicitors (i.e. peptide-,
pathogen-derived signal molecules
oligosaccharide- or lipid-based signalling molecules) that
The resistance gene Pto
originate from the pathogen or represent degradation
The resistance gene product Pto, a serine/threonine
products of the plant cell wall. In gene-for-gene
protein kinase, confers race-specific resistance to strains of
plant–pathogen interactions, these race-specific elicitors
Pseudomonas syringae pv. tomato (the causal agent of bac-
are encoded by pathogen avirulence (avr) genes and their
terial speck disease) that carry the corresponding avirulence
specific recognition is conferred by corresponding plant
gene avrPto. Since the isolation of Pto by map-based
disease resistance (R) genes. R-gene products function extra-
cloning , the Pto–AvrPto interaction has become one of
cellularly or intracellularly. In the case of nonrace-specific
the best-studied model systems for Avr recognition, signal
elicitors, high-affinity binding receptors located in the
initiation and induction of defence responses [4•]. Cell signalling and gene regulation Nonhost resistance Gene-for-gene resistance
Race-specific elicitor /Avirulence gene product
Signal perception Downstream signalling events
Ion fluxes (Ca2+, H+ influx, Cl–, K+ efflux)
PR proteins (glucanases, chitinases)Antimicrobial compounds (phytoalexins)Cell wall fortification
Local resistance Systemic resistance (secondary sites, long lasting)
Overview of plant defence responses after pathogen attack. An
additional protein partners to the receptor or R-gene product,
invading pathogen is perceived by plant membrane-bound receptors
downstream signalling events become initiated, leading to changes in
or extracellular and intracellular R-gene products, that recognise
gene expression and the activation of defence mechanisms.
nonrace-specific or race-specific elicitors as signal molecules.
By mounting a local and systemic resistance, pathogen invasion is
Subsequently, via so far unknown mechanisms that may recruit
arrested. JA, jasmonic acid; SA, salicylic acid.
Recognition of the AvrPto occurs within the plant cell;
determinants, but replacement at Thr204 or Tyr207 by
co-expression of Pto and AvrPto using an Agrobacterium
aspartate resulted in Pto mutants that constitutively
tumefaciens-mediated transient assay in either tomato or
activated an HR in the absence of AvrPto . This gain-
Nicotiana benthamiana caused a hypersensitive response
of-function phenotype was dependent on a functional
(HR), and demonstrated that kinase activity of Pto was
Prf, a gene that was genetically identified as required for
required. A direct physical interaction between the AvrPto
resistance in tomato to P. syringae pathovar (pv.) tomato
and Pto was demonstrated in the yeast two-hybrid system.
and sensitivity to the insecticide fenthion. Whether
The combination of both methods allowed a detailed
(auto)phosphorylation of Pto occurs in vivo, whether an
structure–function analysis of Pto variants generated by
upstream kinase is involved, and whether phosphoryla-
site-directed mutagenesis and domain swapping.
tion is required to increase Pto kinase activity towards itsin vivo phosphorylation targets remain to be addressed.
The Pto activation mechanism was addressed by apply-ing phosphopeptide analysis by matrix-assisted laser
Pto-interacting (Pti) and AvrPto-dependent Pto-interacting
desorption ionisation–time of flight mass spectrometry
proteins (Adi) were isolated from yeast interaction-cloning
(MALDI–TOF/MS), which revealed Thr38 and Ser198
screens . Among these is Pti1, a serine/threonine kinase,
as the major in vitro autophosphorylation sites [6•]. An
whose site of phosphorylation by Pto has been charac-
Ala substitution at these sites rendered the kinase
terised . The involvement of Pti1 suggests the presence
unable to induce HR in the transient expression system
of a signal-amplifying kinase cascade. Pti4, a transcription
[6•]. In a previous publication, the activation segment of
factor that binds to GCC-box cis elements of pathogenesis-
Pto kinase was not only shown to contain AvrPto-binding
related (PR) genes, and is phosphorylated and thereby
Protein kinases in the plant defence response Romeis 409 Signal perception Signal transmission Defence gene activation Macroscopic response
Protein kinases involved in defence signalling. Protein kinases
biochemically activated or has been shown genetically to be required for
(PK superscript) were identified in the context of nonrace-specific
the response. Dotted lines correlate with a negative regulatory role.
elicitation (rectangular-receptor–square-elicitor pair), gene-for-gene
CDPK and MAP kinase cascade components within grey boxes are
interaction (elliptic R-gene product–circular avr product pair), or
members of multigene families, and orthologous genes may fulfil similar
resistance to virulent pathogens, at different signalling levels in different
roles in different plant–pathogen systems. The objective in protein kinase
systems including tobacco (shown in red), tomato (orange), alfalfa (blue),
research is now to find the missing links and characterise the role of
parsley (yellow), rice (brown) and Arabidopsis (green). This scheme
orthologous genes. Future work will also have to integrate protein
shows protein kinases mentioned in this review that are functionally
kinases with other defence signalling mediators such as calcium, reactive
connected with either upstream components or downstream responses.
oxygen species or lipids (see Figure 1), as well as compare responses to
Solid lines indicate a positive regulatory role in which a kinase becomes
pathogen attack with environmental and/or developmental processes.
activated by Pto . Whether these proteins represent
after incubation with the incompatible X. oryzae pv. oryzae
true in vivo Pto kinase targets that are required to establish
strain. Remarkably, the substitution of the extracellular
resistance in tomato to incompatible P. syringae strains
leucine matrix-rich repeat (LRR) and transmembrane
spanning domains from Xa21 with the correspondingdomains from the Arabidopsis receptor kinase BRI1
The resistance gene Xa21
yielded a chimeric receptor that induced these defence
Xa21, a membrane-bound receptor-like kinase (RLK) with
responses after treatment with brassinolide [12•]. The
serine/threonine specificity, confers resistance to bacterial
expression of a kinase-inactive mutant, created by point
leaf blight in rice . Xa21-mediated resistance conforms
mutation by amino-acid exchange in the kinase domain,
a gene-for-gene interaction in which Xa21 expressing
rendered the chimeric receptor kinase inactive. This
plants are resistant to Xanthomonas oryzae pv. oryzae race 6
experimental system will therefore not only allow the
strain, coding for a corresponding, yet uncharacterised, Avr
characterisation of postulated (extracellular) ligand–LRR
product. When expressed in rice cell cultures, Xa21
interactions, but may become useful for the functional
retained its recognition specificity and its functionality in
analysis of the intracellular kinase domain and its role in
inducing defence responses such as H2O2 production,
Xa21-mediated defence signalling, despite not having
changes in gene expression, and induction of cell death
Cell signalling and gene regulation Perception of nonrace-specific elicitation via FLS2
reported upon nonrace-specific elicitation with chitin frag-
Flagellin and its derived peptides were recently shown to
ments , whereas a 55-kDa calcium-stimulated protein
function as nonrace-specific bacterial elicitors of defence
kinase, likely to be a CDPK, was biochemically charac-
responses in cell cultures and plants of different species
terised from French bean cells treated with elicitor
[13,14]. FLS2 codes for a RLK similar to Xa21. Arabidopsis
preparations from the fungus Colletotrichum lindemuthianum
flagellin-insensitive mutant lines were identified by the
. Using tobacco cells that express the Cf-9 resistance
ability of seedlings to grow on flagellin-derived peptides,
gene from tomato as a transgene, a 68/70-kDa CDPK was
and the FLS2 gene was isolated by map-based cloning
biochemically identified by its activation upon elicitation
[15•]. Kinase activity of the intracellular serine/threonine
with the corresponding fungal-derived avirulence gene
kinase domain, as well as the extracellular LRR domain of
product, Avr9 [23•]. Interestingly, enzyme activation was
the FLS2 protein, was shown to be required for flagellin
accompanied by a phosphorylation-dependent transition
binding and signalling . Overexpression of a kinase-
of the CDPK from a non-elicited into an elicited enzyme
associated protein phosphatase (KAPP) rendered plants
form, which could be visualised as a shift in electrophoretic
mobility in immunoblot and in-gel kinase assays.
Interestingly, a second genetically linked locus, FLS1, has
Silencing of a CDPK subfamily using virus-induced gene
been identified and confers insensitivity towards the
silencing (VIGS) resulted in plants that could no longer
flagellin-induced growth inhibition [14,16]. How FLS2
induce a Cf/Avr-specific HR, suggesting that these iso-
and FLS1 participate to constitute the flagellin receptor
forms were required for the activation of the plant defence
complex remains to be shown. Because flagellin induces
(T Romeis, unpublished data). VIGS is a homology-based
responses in different plant species, it may represent an
reverse genetics approach that silences closely related
ideal model system for the study of signal recognition and
genes . VIGS and the recently introduced RNA inter-
transmission, which will allow us to determine shared
ference (RNAi) technology  may prove important
and distinct components between R-gene-dependent and
reverse genetics tools that could be used when gene families
nonrace-specific elicitation signalling.
are studied and entire subfamilies have to be targeted. Protein kinases functioning downstream MAP kinases of the recognition of the pathogen/
Mitogen-activated protein kinases (MAPKs) from several
plant species were shown to be activated during plant
In vivo labelling experiments, as well as studies with
responses to elicitors or pathogens (see review by J Sheen,
non-specific pharmacological inhibitors, provided early
pp 392–400) [3,26]. Two groups of orthologous MAP
evidence that protein kinases participate in downstream
kinase genes comprising WIPK, SIMK, AtMPK3, and
signalling to activate plant defence responses [2,4•,17,18]. ERMK from tobacco, alfalfa, Arabidopsis, and parsley,
Several gene-for-gene and nonrace-specific elicitation
respectively, and SIPK, SAMK, and AtMPK6 from tobacco,
systems are currently employed to study these downstream
alfalfa and Arabidopsis have been characterised [27–31].
signalling events and, in the majority, recognition of the
MAP kinase signalling is complex: gene families exist for
elicitor/avirulence product occurs extracellularly. This
each of the MAPKKK, MAPKK, and MAPK members of
offers the advantage of an amenable experimental system,
the phosphorylation cascade. The enzymes are multi-func-
cells or plants, in which the elicitor can simply be added
tional and specific isoforms become activated by both
externally and kinase activation can be studied over a time
race- and nonrace-specific pathogen-related elicitation as
course. So far, only a few protein kinases have been
well as by environmental stimuli [3,32]; and several MAP
analysed in the biological context of viral and bacterial
kinase pathways are utilised in parallel upon a single
pathogen attack (e.g. tobacco N–tobacco mosaic virus, and
elicitation stimulus [30,33,34]. MAP kinases were shown
Arabidopsis RPS5–P. syringae gene-for-gene interactions).
biochemically to be activated upon elicitation, suggesting
Some downstream kinases have now been identified by
a positive regulatory role in defence signalling. However, a
biochemical methods and their genes cloned.
thorough reverse genetics or mutational analysis is so farlacking. Now, we are beginning to answer many important
Calcium-dependent protein kinases (CDPKs)
questions regarding MAP kinase signalling; for example,
CDPKs comprise a family of plant-specific, multi-func-
are MAP kinases required for defence signalling, which
tional serine/threonine protein kinases in which a
distinct cellular responses do they mediate, and how can
regulatory calcium-binding domain is directly linked to the
stimulus and pathway specificity be accomplished?
kinase domain [19,20]. Elicitor-induced calcium influx andprotein kinase activity have been reported from many
By applying specific pharmacological inhibitors that
pathosystems as one of the earliest responses required for
interfere with MAP kinase signalling, MAP kinase
further downstream signalling. CDPKs are therefore ideally
activation (analysed by in-gel kinase activity) was shown
structured for sensing changes in intracellular calcium con-
to correlate with the induction of defence-gene expres-
centration and translating them into kinase activity.
sion and HR. Inhibition of MAP kinase pathways,
Transcript accumulation of NtCDPK1 from tobacco was
demonstrated by lack of SIPK kinase activity, affected
Protein kinases in the plant defence response Romeis 411
elicitor-induced accumulation of HIN1 transcript in
interferes with some gene-for-gene interaction systems,
tobacco cells treated with harpin from P. syringae pv. phase-
and defence responses are already activated due to proto-
olicola . In analogous experiments, the HR cell death
plasting and/or protoplasts do not respond to elicitation.
response in tobacco and Arabidopsis cells was compro-
Therefore, transient expression experiments in leaves may
mised upon exposure to fungal elicitors from Trichoderma
be exploited as an alternative system. viride  or Phytophthora parasitica , and harpin fromP. syringae pv. syringae .
Are MAP kinases required for the plant defence? In bio-chemical approaches, protein kinases were identified that
A significant step in further understanding MAP kinase
become activated upon certain stimuli but reverse genetics
signalling was accomplished in elegant studies in which a
and mutational analysis have now yielded the first two well-
dexamethasone-inducible, constitutively active gain-of-
defined loss-of-function mutations in MAP kinase cascade
function mutant of a tobacco MAPK kinase, NtMEK2, was
components that are related to the plant defence response.
generated and transiently expressed in tobacco leaves.
An Arabidopsis line that carried a modified maize Ds trans-
Dexamethasone application resulted in HR-like cell death
poson element in the AtMPK4 gene was identified [44•].
accompanied by the induction of defence-related genes,
The mpk4 mutant line has a dwarf phenotype, showed
both of these processes were preceded by an increase in
enhanced resistance to virulent bacterial (P. syringae) and
endogenous WIPK and SIPK kinase activity [39••]. In
fungal (P. parasitica) pathogens, exhibited constitutive SAR
addition, steroid-inducible expression of SIPK alone and
and defence-related gene expression, but lacked jasmonic
elevation of its activity was sufficient to mimic these
acid-dependent gene induction. These responses were
responses (S Zhang, Y Liu, personal communication). Such
dependent on MPK4 kinase activity, as a kinase inactive
experiments carry the risk that the ectopic expression of
mutant allele failed to complement the mpk4 mutation.
constitutively active or dominant kinase variants may
Thus, the potential kinase cascade utilising MPK4 apparently
result in the phosphorylation of non-physiological target
has a negative regulatory role in the plant defence.
proteins. Also, the overexpression of an activated kinase mayinterfere with its normal intracellular localization, its recog-
In addition, the Arabidopsis edr1 mutant, isolated from a
nition specificity, or its proper recruitment of components
genetic screen, displayed enhanced resistance against the
into protein complexes. Nevertheless, they provide a
usually virulent bacterial strain P. syringae and the fungal
powerful tool that may become useful for the characterisation
powdery mildew pathogen Erysiphe cichoracearum .
of immediate downstream signalling responses. EDR1 codes for a MAPKK kinase. The recessive nature ofthe mutation suggests that EDR1, like MPK4, may function
The identification of NtMEK2 but not other MAPK kinases
at the top of a MAP kinase cascade that negatively regulates
participating in the activation of defence responses suggests
that distinct (multi-functional) components are recruitedinto signalling cascades depending on the inducing stim-
How the negative regulatory role of the genetically identi-
ulus [39••]. Signal- and/or MAP-kinase-specific upstream
fied MAPK cascade components EDR1 and MPK4 fits
members of MAPK kinases (MEKs) have also been found
into the defence signalling picture, together with the
in other plant systems [40,41•,42]. A mechanism has
positive regulatory role for MPK3 and MPK6 orthologues,
recently been proposed to demonstrate how signal speci-
remains to be shown. Meanwhile, alternative interpreta-
ficity could be acquired: the composition of protein
tions taking into account the potential assembly of protein
complexes that sense signals varies depending on the
complexes and crosstalk between different signalling
incoming signal, and these protein complexes might be
assembled with the help of scaffold proteins [26,43].
Recently, transient transformation of cell or leaf protoplasts
Forward-genetic screens designed for suppressors of
has been established enabling the expression of epitope-
R-gene-dependent or nonrace-specific signalling have so
tagged kinase variants with a reporter construct to study
far yielded astonishingly few mutations in signal transduc-
their effect on transcription. This elegant system allows
tion components, given the number of different systems
the cell-based reconstitution of entire signalling cascades
and laboratories that have pursued this approach. The lack
and has already been successfully employed to define an
of such knock-out mutations is explained by potential
oxidative-stress activated MAP kinase cascade [41•]. One
lethality or functional redundancy. One exception has now
can envisage investigating protein kinases by combining
been reported from a screen for loss of RPS5-mediated
such a protoplast system with nonrace-specific elicitors.
resistance in Arabidopsis to P. syringae strains containing the
Such a system has been established in the interaction of
avirulence gene AvrPphB, which resulted in a recessive
parsley protoplasts with the Pep13 elicitor, a glycoprotein-
mutation in PBS1 . PBS1 encodes a serine/threonine
derived peptide from Phytophthora sojae (H Hirt, personal
kinase that belongs to a novel protein kinase subfamily
communication), as well as in the Arabidopsis–flagellin
[49•], and the pbs1-2 mutant allele, which has a Gly to Arg
system (J Sheen, personal communication). However, the
substitution in the kinase activation segment, suggests
generation of protoplasts from leaves or cell cultures
that kinase activity is required for resistance. Whether PBS1
Cell signalling and gene regulation
participates directly in the Avr–R recognition process, for
References and recommended reading
example by phosphorylating one of these components, or
Papers of particular interest, published within the annual period of review,have been highlighted as:
represents a downstream signalling intermediate, remainsto be addressed. However, PBS1 is not required for the
function of the related RPS2 and RPM1 R-genes.
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binding in the yeast two-hybrid system and hypersensitive response-inducingactivity using an A. tumefaciens-mediated transient assay in the presence of
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The transient expression of epitope-tagged, constitutively active kinase variants
forms, visualised as shift in electrophoretic mobility in in-gel kinase assay and
in protoplasts represents an excellent tool to reconstitute phosphorylation
immunoblot. Because of that shift, caused by phosphorylation, the activation
cascades and address their function in response to stress stimuli.
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A rare example in which a genetic screen for enhanced disease resistanceyielded a MAP kinase cascade component. The manifestation of the edr1-
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associated resistance phenotype is dependent on salicylic acid production. Avr9- and Cf-9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor,
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Protein kinases induced by osmotic stresses and elicitor
A discussion stimulating commentary on the recently characterised
molecules in tobacco cell suspensions: two crossroad MAP Arabidopsis edr1 and mpk4 mutants, which offers an alternative interpretation
kinases and one osmoregulation-specific protein kinase. FEBS
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these MAP kinase cascade components from the angle of multi-proteincomplexes involved in crosstalk between signalling pathways.
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The first example in which a genetic screen designed for loss of R-gene-
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mediated resistance (RPS5) to an incompatible pathogen (P. syringaep47 protein kinase during hypersensitive cell death in a culture of
carrying AvrPphB) yielded a protein kinase that represents a potentially early
tobacco cells. Plant Physiol 1999, 119:1465-1472.
component in downstream signal transduction. Cell signalling and gene regulation
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Lecourieux-Ouaked F, Pugin A, Lebrun-Garcia A: Phosphoproteins
Nahreini TS, Resing KA, Ahn NG: Identification of novel involved in the signal transduction of cryptogein, an elicitor of defense MAP kinase pathway signaling targets by functional reactions in tobacco. Mol Plant Microbe Interact 2000, 13:821-829. proteomics and mass spectrometry. Mol Cell 2000, 6:1343-1354.
52. Peck SC, Nühse TS, Hess D, Iglesias A, Meins F, Boller T: Directed
Changes in the protein pattern were analysed either by 2D-gel elec-
proteomics identifies a plant-specific protein rapidly
trophoresis, after stimulation of a human cell line with phorbolester in the
phosphorylated in response to bacterial and fungal elicitors. Plant
absence or presence of pharmaceutical inhibitors of MAP kinase signalling,
Cell 2001, 13:1467-1475.
or in a gain-of function approach, by expressing a constitutively active MAPK
The effect of flagellin elicitation on Arabidopsis in the presence of radioac-
kinase. Proteins were identified by mass spectrometry. These changes rep-
tively labelled orthophosphate was analysed for rapid changes in the phos-
resent predominantly post-translational modifications of proteins, whose cor-
phoprotein pattern by 2D-gel electrophoresis and mass spectrometry. This
responding genes were not detected by DNA-microarray analysis in the
approach will become instrumental in the kinase field for both phosphorylation
target identification and kinase regulation.
Binary Volumetric Octree Representation for Image-Based Rendering Using single BVO structure for both geometry and color data Abstract increasing memory requirements with growing like n , but A Binary Volumetric Octree (BVO) is a volume array with binary preserve volumetric structure. In contrast to Binary Space-opacity voxels, represented as octree. The BVO structure allows Par
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