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
Pii: s1369-5266(00)00193-xProtein kinases in the plant defence response
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 Addresses
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: firstname.lastname@example.org 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: email@example.com 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 Abbreviations
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 R
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 Introduction
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
Race-specific elicitor /Avirulence gene product Signal perception
Downstream signalling events
Ion fluxes (Ca2+, H+ influx, Cl–, K+ efflux) Defence mechanisms
PR proteins (glucanases, chitinases)Antimicrobial compounds (phytoalexins)Cell wall fortification Local 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
Defence gene activation
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
of the recognition of the pathogen/
Mitogen-activated protein kinases (MAPKs) from several pathogen-derived signal
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
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