Resistance proteins: molecular switches of plant defence

Resistance proteins: molecular switches of plant defenceFrank LW Takken1, Mario Albrecht2 and Wladimir IL Tameling3

Specificity of the plant innate immune system is often

conferred by resistance (R) proteins. Most R proteins contain

leucine-rich repeats (LRRs), a central nucleotide-binding site

(NBS) and a variable amino-terminal domain. The LRRs are

mainly involved in recognition, whereas the amino-terminal

domain determines signalling specificity. The NBS forms part of

a nucleotide binding (NB)-ARC domain that presumably

functions as a molecular switch. The conserved nature

of NB-ARC proteins makes it possible to map mutations

of R protein residues onto the crystal structures of related

NB-ARC proteins, providing hypotheses for the functional roles

of these residues. A functional model emerges in which the

LRRs control the molecular state of the NB-ARC domain.

Pathogen recognition triggers nucleotide-dependent

conformational changes that might induce oligomerisation,

thereby providing a scaffold for activation of downstream

signalling components.

Addresses1 Plant Pathology, Swammerdam Institute for Life Sciences, University of

Amsterdam, PO Box 94062, 1090 GB Amsterdam, The Netherlands2 Max Planck Institute for Informatics, Stuhlsatzenhausweg 85, 66123

Saarbrucken, Germany3 The Sainsbury Laboratory, John Innes Centre, Norwich Research Park,

Colney, Norwich NR4 7UH, UK

Corresponding author: Takken, Frank LW ([email protected])

Current Opinion in Plant Biology 2006, 9:383–390

This review comes from a themed issue on

Biotic interactions

Edited by Anne Osbourn and Sheng Yang He

Available online 19th May 2006

1369-5266/$ – see front matter

# 2006 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2006.05.009

IntroductionTo combat pathogens, plants have evolved a sophisti-

cated, multilayered system of passive and active

defence mechanisms. Except for the RNA-based anti-

viral defence, which is a form of adaptive immunity,

plants rely on an innate immune system [1�,2]. Microbes

disclose their presence to the host’s innate immune

system through pathogen-associated molecular patterns

(PAMPs). These PAMPs are evolutionary conserved

molecules that are often indispensable for the microbe’s

lifestyle [2]. They act as general elicitors that are recog-

nized by the host via specialized PAMP receptors,

triggering subsequent induction of ‘basal’ defence

responses [2,3].

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Many pathogens, however, have found ways to evade and/

or actively suppress the host’s basal defence responses,

and hence have acquired the ability to cause plant dis-

eases. To evade recognition, a pathogen might hide or

alter a specific PAMP. For instance, Agrobacterium tume-faciens has a modified flagellin domain that can no longer

be recognized by the Arabidopsis flagellin receptor [4].

Alternatively, pathogens might suppress basal defence

responses using specific effector molecules [5]. One of the

best-studied examples is the suppression of RIN4-regu-

lated basal defence in Arabidopsis by the Pseudomonassyringae effector proteins AvrRpm1 and AvrRpt2 [6��,7]. A

drawback for the pathogen is that, by manipulating the

host cell machinery, it risks triggering the activation of a

second line of plant defence, called ‘gene-for-gene’ resis-

tance. This type of resistance is based on the presence of

specific resistance (R) genes that mediate the recognition

of race-specific effectors (Avr proteins). The emerging

picture is that R proteins monitor the integrity of com-

ponents of the basal PAMP-recognition-based immune

system. Perturbation of these components by effector/Avr

proteins could trigger activation of the R protein. To

elaborate on the RIN4 example given above, this Avr

target, which plays a role in regulating basal defence, is

‘guarded’ by at least two different R proteins: RPM1 and

RPS2. Phosphorylation of RIN4 in the presence of

AvrRpm1 triggers RPM1 activation, whereas degradation

of the RIN4 protein by AvRpt2 activates RPS2 [8–10]. In

the latter case, AvrRpt2-mediated cleavage of RIN4

releases its association with RPS2, and thereby removes

the negative regulation of RPS2 function by RIN4 [11].

Release of negative regulation triggers R-protein activa-

tion, which results in the induction of rapid defence

responses, frequently culminating in a hypersensitive

response (recently reviewed in [1�]). These examples

provide a mechanistic link between basal and gene-for-

gene resistance. If guarding the components of PAMP

signalling pathways turns out to be the main function of R

proteins [2,12�], it would provide the plant with a robust

immune system [6��].

Numerous R proteins have been identified and the

majority contain a nucleotide binding site (NBS) and a

carboxy-terminal leucine-rich repeat (LRR) domain [13].

At the N-terminus, NBS-LRR proteins carry either a

coiled coil (CC) domain (in CNL proteins) or a domain

that has homology to the Toll/Interleukin-1 Receptor

(TIR) domain (in TNL proteins) [14]. The N-terminal

domain is thought to be involved in downstream signal-

ling, whereas the LRRs seem to be the main determinant

for recognition specificity [1�,13]. The NBS is part

of a larger domain, which is called nucleotide binding


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http://dx.doi.org/10.1016/j.pbi.2006.05.009

384 Biotic interactions

Figure 1


(NB)-ARC because it is shared between R proteins and

the apoptotic regulators human apoptotic protease-acti-

vating factor 1 (APAF-1) and its Caenorhabditis eleganshomolog CED-4 [15]. Proteins that have an NB-ARC

domain are evolutionary related to the mammalian

NACHT-LRR (NAIP, CIITA, HET-E, TP1) proteins

(NLRs), many of which also function in innate immunity

[16,17,18�]. Both NB-ARC and NLR proteins belong to

the STAND (signal transduction ATPases with numer-

ous domains) family of NTPases [18�]. The nucleotide-

binding domain of these proteins is proposed to work as

an NTP-hydrolyzing switch, regulating signal transduc-

tion by conformational changes [18�].

In this review, we focus on the regulatory role of the NB-

ARC domain in NBS-LRR proteins and its proposed

function as a molecular switch. Since no three-dimen-

sional (3D) structures are available for NBS-LRR pro-

teins, comparisons are made with two metazoan NB-ARC

proteins whose crystal structures have recently been

solved, APAF-1 and CED-4 [19��,20�]. To provide a link

between structure and function, known autoactivating

and loss-of-function mutations in the NB-ARC domain

of NBS-LRR proteins (Figure 1 and Supplementarytable) are mapped onto the 3D structures of APAF-1

and CED-4 (Figure 2). In agreement with recent models

[21], NBS-LRR proteins are proposed to work as dynamic

signalling molecules, performing reversible intra- and

intermolecular interactions. The outcome of these inter-

actions determines whether a plant activates its defence

responses.

Structural features of the NB-ARC domainAs suggested previously, the APAF-1 and CED-4 crystal

structures [19��,20�] are highly similar to that of AAA+

Architecture of the NB-ARC1-ARC2 domains. (a) General protein

topology of the NB-ARC1-ARC2 domains as derived from the APAF-1

crystal structure (cylinders and arrows represent a-helices and b-

strands, respectively) [19��]. The central b-sheet in the NB domain is

formed by five b-strands (cyan), which adopt a 2-3-4-1-5 topology, and

is surrounded by seven a-helices (red). The b-strands b1 and b3

encompass the Walker A (orange line) and Walker B motifs, respectively.

The ARC1 and ARC2 domains are shown in purple and dark blue,

respectively. (b) Mutations of the R proteins I-2, N, L6, Prf, RPM1, RPS2,

Rx, SSI4 are mapped onto the aligned NB-ARC1-ARC2 domain

structures (red, purple and blue boxes) of APAF-1 and CED-4. Sequence

motifs that have been described in other publications are annotated (see

also Table 1). Autoactivating mutations are highlighted in green, loss-of-

function mutations in yellow. Mutations and the corresponding sequence

positions of APAF-1 (CED-4) are linked by horizontal lines. Black-

coloured text in orange boxes indicates nucleotide-binding amino acids

in APAF-1/CED-4 [19��,20�]. Orange-coloured sequence positions of

APAF-1/CED-4 represent amino acids that form part of the active site

and thus might affect nucleotide binding and hydrolysis, even though

they have not been reported to be involved directly in nucleotide binding.

The locations of a-helices and b-strands (red cylinders and blue arrows,

respectively) are derived from the crystal structure of APAF-1 [19��]. All

mutations together with literature references are listed in the

Supplementary table, which additionally maps RPM1 loss-of-function

mutations that were not found in specific sequence motifs.


Resistance proteins Takken, Albrecht and Tameling 385

Figure 2

Different conformations of the NB-ARC domain structures of ADP-binding APAF-1 (right) and ATP + Mg-binding CED-4 (left). The a-helices and

b-strands of NB are shown in red and cyan, respectively, whereas ARC1 and ARC2 are shown in purple and dark blue, respectively. The b-strands

of NB are numbered as in Figure 1. APAF-1 (CED-4) positions that are mapped to autoactivating and loss-of-function mutations of R

proteins are highlighted in green and yellow, respectively (Figure 1b). In the Walker A motif, only K160 (K165) is annotated, the locations of the

remaining loss-of-function mutations are shown by the yellow-coloured P-loop. The putative sensor-I arginine (in the RNBS-B motif) is indicated

by an orange text box. The amino- and carboxy-termini of APAF-1 and CED-4 are also marked. ATP, ADP, and Mg atoms are depicted as balls-and-

sticks model.

proteins [18�]. Alignment of the NB-ARC domains of

NBS-LRR proteins with those of APAF-1 or CED-4

makes it possible to map the locations of R protein

mutations, as well as conserved sequence motifs, onto

the crystal structures (Figures 1b and 2, Supplementarytable; [16,22]).

The NB-ARC domain of APAF-1 actually consists of four

subdomains: the nucleotide-binding subdomain (NB)

and three ARC subdomains (ARC1–ARC3) [19��]. The

NB subdomain is a ‘classical’ NTPase fold, which places

NB-ARC proteins within the large group of P-loop


NTPases [23]. This fold forms a parallel b-sheet flanked

by a-helices. The b-sheet assumes a 2-3-4-1-5 topology,

where b-strand 1 is placed between b-strands 4 and 5

(Figures 1a and 2). In the b-sheet, the strands b1 and b3

are associated with the P-loop and Walker B motif,

respectively (Figures 1a and 2). ARC1 consists of a

four-helix bundle, ARC2 adopts a winged-helix fold,

and ARC3 also forms a helical bundle [18�,19��,20�].Although the first two ARC domains of APAF-1 are

conserved in NBS-LRR proteins, ARC3 is absent

(Figure 1; [16]). Instead, ARC2 is connected to the

LRR domain by a short linker [24]. The nucleotide is



Table 1

Motifs and synonymous names annotated by different authors in the NB-ARC domain.

Motif [18�]a Synonym [24,25] Synonym [14] Synonym [15] Synonym [42]

hhGRExEb – – – –

Walker A P-loop Motif I Kinase 1a Motif I

– RNBS-A Motif II – Motif II

Walker B Kinase 2 Motif III Kinase 2 Motif III

hhhToR RNBS-B Motif IV Kinase 3a Motif IV

– RNBS-C Motif V Motif 4 –

GxP GLPL Motif VI Motif 3 Motif V

– – Motif VII – –

– – Motif VIII Motif 2 –

– RNBS-D Motif IX – –

– – Motif X – –

hxhHD MHDV Motif XI Motif 5 –

a Citation numbers refer to the references naming the motifs.b Names indicated in bold have been used in the figures and throughout the text.

bound at the interface between the NB, ARC1 and ARC2

subdomains. Depending on the type of the bound nucleo-

tide (ATP or ADP), however, very different conforma-

tional states are adopted by the APAF-1 and CED-4

domain structures (Figure 2).

Conserved motifs in NBS-LRR proteinsinvolved in nucleotide bindingSeveral conserved sequence motifs have been annotated

in NBS-LRR proteins (Table 1; [14,15,24,25]) and, not

surprisingly, many residues within these motifs are

located at key positions in the 3D structure (Figures

1b and 2). The most-conserved motifs in P-loop NTPases

are the P-loop itself (the Walker A motif) and the Walker

B motif. The P-loop has the consensus sequence

GxxxxGKS/T (where x indicates any residue), in which

the lysine (K) binds the b- and g-phosphates and S/T

binds a Mg2+ ion. The functional importance of these

motifs can be appreciated from the many identified loss-

of-function mutations (Figure 1b). Mutation of the con-

served lysine in the P-loop generally causes reduced ATP

binding, which was also observed for the NBS-LRR

protein I-2 [26]. In the Walker B consensus hhhhDD/E

(where h is mostly a hydrophobic residue), the invariant

aspartate (D) is important for the indirect coordination of

the Mg2+ ion via a water molecule, whereas the second

acidic residue is thought to be a catalytic base that is

required for ATP hydrolysis [18�,27]. The importance of

the Walker B motif for the function of the NBS-LRR

proteins N, RPS2 and Rx has also been confirmed by

mutational analysis (Figures 1 and 2). ATPase activity of

NB-ARC proteins was shown for APAF-1 [28��] and two

NBS-LRR proteins (I-2 and Mi-1) [26]. The involvement

of the second aspartate of the Walker B motif in hydro-

lysis is underscored by the finding that the D283E muta-

tion in I-2 affects ATP hydrolysis but not binding [29��].

Near the N-terminus of the a-helix that precedes the

P-loop lies the STAND-characteristic motif, hhGRExE


[18�]. In APAF-1, the arginine (R) in this motif forms a

hydrogen bond with the base of ADP via a water

molecule. Mutation of the glycine (G174R) in RPM1

leads to loss-of-function, possibly caused by defective

nucleotide binding. Besides arginine, the four preced-

ing residues are also responsible for nucleotide binding

in APAF-1. One of them, V127 of APAF-1, dictates

adenine specificity over guanine specificity. This is in

line with the specificity for adenine of the NBS-LRR

protein I-2 [26]. The hhGRExE motif belongs to a

linker region between the N-terminal domain and the

NB subdomain. In APAF-1 and CED-4, it connects the

N-terminal effector caspase recruitment domain

(CARD) with the NB-subdomain, and upon protein

activation this linker region might propagate conforma-

tional changes that result in the exposure of the effector

domain for binding with downstream interaction part-

ners [30].

The hhhhToR signature (o designates an alcoholic resi-

due) (known as the RNBS-B motif) in strand b4 of the NB

subdomain, corresponds to the sensor I motif in AAA+

proteins [31]. The arginine ‘senses’ the presence of the

g–phosphate and relays this information to other parts of

the protein [32]. A putative role for this arginine as a

g-phosphate sensor is supported by the fact that it inter-

acts with the g-phosphate in the CED-4 crystal structure

[20�]. Functional importance of this motif in NBS-LRR

proteins (N, RPM1 and Prf) was shown by mutational

analysis (Figures 1b and 2), but its predicted role as a

g-phosphate sensor awaits exploration.

Although an autoactivating mutation S233F of I-2 was

reported in the RNBS-A motif [25] close to the active site

[29��], further loss-of-function mutations in NBS-LRR

proteins were identified in the RNBS-C motif [15,25], in

the GxP (GLPL) motif and in motif VII [14,18�,25], all of

which contain amino acids that are involved in ATP

binding (Figures 1b and 2).



In the loop that connects ARC1 with ARC2, two residues

in CED-4 are involved in ATP binding. The autoactivat-

ing mutation G422A of SSI4 is located in the subsequent

helix (motif VIII) (Figures 1b and 2). A serine (S) in the

poorly conserved ‘motif X’ [14] in the ARC2 subdomain

of APAF-1 is also involved in nucleotide binding. This

motif lies within the CARD-binding interface, which

might explain the poor conservation of this motif

amongst NB-ARC proteins. The highly conserved

MHD-motif (hxhHD) is also located in ARC2 [18�].The histidine (H) in this motif directly interacts with

the b-phosphate in APAF-1 (Figures 1b and 2), and might

replace the sensor II motif in AAA+ proteins [27] that

is thought to be involved in nucleotide-dependent

conformational changes [19��]. Strikingly, mutagenesis

of either the histidine or aspartate in several NBS-LRR

proteins results in autoactivation, indicating that these

residues have a crucial role (Figure 1b).

The NB-ARC: a STANDard switch?Biochemical and structural analyses of APAF-1 and CED-4

show that the NB-ARC domain is perfectly suited as a

molecular switch to regulate signalling pathways through

conformational changes [19��,28��]. But what about the

NB-ARC as a regulatory switch in NBS-LRR proteins?

For two NBS-LRR proteins, it has been reported that

changes in intra- and intermolecular interactions upon

Figure 3

Model for the switch function of NBS-LRR proteins. In the absence of a patho

LRR exerts its negative role by stabilizing the ADP-bound state. The presen

conformational change in the NB-ARC domain that allows the release of ADP

the N-terminal effector domain, releasing its signalling potential. The ATPase

protein to its resting state.


Avr perception depend on a functional NB-ARC domain.

The interaction of the CC domain with the NB-ARC-LRR

part of the potato CNL protein Rx is disrupted upon Avr

perception, and this intramolecular interaction requires a

wildtype P-loop [33]. Likewise, oligomerisation of the

tobacco TNL protein N upon co-expression of the Avr

protein requires an intact P-loop [34��]. In both Rx and N, a

double mutation from GK to AA in the P-loop abolished

these interactions. Additional evidence of an important

role for nucleotide binding in NBS-LRR protein function

comes from biochemical analysis of two autoactivating

mutations in I-2. Both mutations are present in the NB

subdomain and were found to affect ATP hydrolysis but

not nucleotide binding [29��].

On the basis of these data, we propose a model that

explains the effect of these mutations and, more impor-

tantly, suggests how the NB-ARC domain might serve as

a molecular switch. The specific reduction of ATP hydro-

lysis in the autoactivating I-2 mutant proteins is thought

to result in the accumulation of the ATP-bound state.

When this state exceeds a certain threshold, it would

trigger defence signalling. Furthermore, in vitro binding

studies on I-2 indicated that the ADP-bound state is

highly stable [29��]. Therefore, this state is probably

the resting state, whereas the ATP-bound state is the

activated form [29��].

gen, an NBS-LRR protein is in the OFF-state (resting state), in which the

ce of an elicitor (Avr) affects the LRR domain, which induces a

. ATP binding subsequently triggers a second conformational change in

activity of the protein attenuates the signalling response and returns the



The LRRs play an important negative role as well as a

positive role in regulating NBS-LRR protein activity.

Negative regulation is suggested by several autoactivat-

ing mutations that were identified in this domain. Dele-

tion of the entire LRR domain does not, however,

normally result in constitutive activity [35–37]. On the

basis of these data, it is possible to conceive a dynamic

model for NBS-LRR protein function, as shown in

Figure 3. The presence of an Avr is recognized by the

LRR domain, which induces a conformational change in

the NB-ARC domain. This allows the exchange of ADP

for ATP. ATP binding then triggers a conformational

change in the N-terminal effector domain, releasing the

NBS-LRR’s signalling potential. Provided that the auto-

activating mutations in I-2 do not alter the inhibitory

effect of the LRR, it is likely that the proposed cycle runs

slowly in the absence of a pathogen (otherwise inhibition

of the ATPase activity would not lead to autoactivation).

Such a continuous turnover would explain why there is a

certain degree of constitutive activity of NBS-LRR pro-

teins in the absence of an Avr, resulting in a fitness

penalty [38]. Furthermore, it would explain why over-

expression of NBS-LRR genes can result in Avr-inde-

pendent defence signalling [39]. It might also explain

why it is of crucial importance for a cell to tightly control

NBS-LRR protein levels [40].

The proposed switch model for NBS-LRR proteins is

similar to the apparent switch mechanism for APAF-1,

in which the (d)ATP state is the active form. In the case of

APAF-1, however, the resting state is also (d)ATP- rather

than ADP-bound, and after APAF-1 activation, a single

round of hydrolysis is induced. Only when the concentra-

tion of (d)ATP in the cell is sufficiently high, will the bound

(d)ADP be exchanged for (d)ATP, inducing a second

conformational change that allows the assembly of the

active signalling complex [28��]. Further studies with

full-length R proteins need to be performed to determine

which nucleotide is bound in the resting complex: ADP as

proposed in the model or ATP as in APAF-1.

Until recently, it was unknown whether NBS-LRR pro-

teins oligomerise upon activation, as do APAF-1 and

many other STAND proteins [18�]. APAF-1 and its

Drosophila and C. elegans orthologues DARK1 and

CED-4 form wheel-like structures after activation, pro-

viding a platform for activation of procaspases

[19��,20�,41]. As mentioned before, Mestre and Baul-

combe [34��] showed Avr-dependent oligomerisation of

the tobacco R protein N via a TIR–TIR interaction.

Additional studies should clarify whether oligomerisation

is a common theme for NBS-LRR protein activation.

Conclusions and future prospectsIn recent years, impressive progress has been made in our

understanding of R-protein function. We have begun to

reveal the mechanistic and biochemical basis of NBS-


LRR protein action, and the role that nucleotide binding

plays in inter- and intramolecular interactions. Our cur-

rent understanding is, however, fragmentary because

such interaction patterns or nucleotide-binding has been

analyzed for only a few NBS-LRR proteins. As exempli-

fied in this review, the new structural view of the NB-

ARC domain supports the formation of hypotheses to

explain how mutations in this domain might affect

nucleotide binding and hydrolysis, and thus the confor-

mation and activity of NBS-LRR proteins. More bio-

chemical and structural studies are now required to

validate these hypotheses.

To gain further understanding of how NBS-LRR proteins

regulate plant defence, the signalling complexes need to

be characterized and their dynamics studied in more

detail. The identification of the interacting partners will

be the next challenge, and several such studies are

currently underway [1�,21]. An important focus for the

future will be the purification of full-length NBS-LRR

proteins. This will allow their in vitro characterization

and, hopefully, the elucidation of their 3D structures,

which will greatly assist the clarification of the molecular

function of these intriguing proteins.

AcknowledgementsM Albrecht is financially supported by the German National GenomeResearch Network (NGFN). This work was also conducted in the contextof the BioSapiens Network of Excellence, funded by the EuropeanCommission under grant number LSHG-CT-2003-503265. We thankMartijn Rep for useful comments on the manuscript.

Appendix A. Supplementary dataSupplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.pbi.2006.

05.009.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest�� of outstanding interest

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An insightful review highlighting differences and similarities betweeninnate immune signalling in plants and mammals. The authors providean up-to-date overview of the current models on how host- and non-hostresistance are correlated. Furthermore, they provide a wealth of informa-tion on R protein function and the interactions that these proteins havewith chaperones and proteins that are involved in targeted proteolysis.

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6.��

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This paper builds further on the work presented in [7,8]. The authors showfirst that both AvrRps2 and AvrRpm1 are able to suppress the induction ofbasal defence responses induced by either a type III secretion mutant ofPseudomonas syringae or the PAMP flg22. Next, they show that of one ofthe targets of these Avr proteins, RIN4, is a negative regulator of basaldefence signalling. As RIN4 is inactivated by both Avr proteins, whichwould result in increased rather than suppressed basal defenceresponses, the authors suggest that a second positive regulator mustbe present, whose function is also suppressed by the Avr proteins. Themain conclusion is that Avr proteins perturb essential components of thebasal defence system, and that this perturbation is sensed by host-specific resistance proteins, thereby elegantly linking both defence sys-tems.

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18.�

Leipe DD, Koonin EV, Aravind L: STAND, a class of P-loopNTPases including animal and plant regulators ofprogrammed cell death: multiple, complex domainarchitectures, unusual phyletic patterns, and evolution byhorizontal gene transfer. J Mol Biol 2004, 343:1-28.

A new class of P-loop NTPases is defined: the STAND (signal transduc-tion ATPases with numerous domains) proteins. The authors identifiedSTAND proteins in a wide range of organisms ranging from archae andbacteria to mammals, fungi and plants. Several protein clades were


detected, and although the central NTPase domain is strongly conservedin evolution, a large variability in the type of adjoining domains isapparent in STAND proteins. These multidomain proteins are involvedin a plethora of signalling processes. Besides the NTPase domain, theseproteins often contain DNA- or protein-binding domains and sometimesenzymatic domains. In analogy to AAA+ ATPases, the authors proposethat ARC1 functions as a lever to transmit conformational changes thatare induced by NTP hydrolysis to N-terminal effector domains. A uniquefeature of this class of STAND proteins is that they seem to combineadaptor, switching, and scaffolding function into a single protein.The assembled sequence alignments also clearly indicate that theNB-ARC1-ARC2 domains in NB-ARC and NLR proteins have a commonorigin.

19.��

Riedl SJ, Li W, Chao Y, Schwarzenbacher R, Shi Y: Structure ofthe apoptotic protease-activating factor 1 bound to ADP.Nature 2005, 434:926-933.

This paper describes the crystal structure of the NB-ARC protein APAF-1bound to ADP. Here, ADP seems to function as an organizing centre,which maintains the specific structure of the four identified subdomainsNB-ARC1-ARC2-ARC3. This paper provides important insight into thestructure of the NB-ARC domain and the functional role of variousresidues in interdomain interactions and nucleotide binding.

20.�

Yan N, Chai J, Lee ES, Gu L, Liu Q, He J, Wu JW, Kokel D,Li H, Hao Q et al.: Structure of the CED-4–CED-9 complexprovides insights into programmed cell death inCaenorhabditis elegans. Nature 2005, 437:831-837.

The authors of this paper describe the crystal structure of the NB-ARCprotein CED-4 of Caenorhabditis elegans. They present the structure ofthe asymmetric CED-4 dimer bound to the inhibitor protein CED-9.Furthermore, they show that the apoptosis-inducing protein EGL-1 cancompete with CED-4 for CED-9 binding. To this end, the CED-4 dimer isreleased and oligomerises in a tetramer, which leads to autoactivation ofthe CED-3 killer protease. Interestingly, in both the inactive dimer and theactivated tetramer, the NB-ARC domain is associated with ATP. No invitro ATPase activity could be detected. So, it remains unclear if CED-4 iscapable of, or indeed requires, hydrolysis of ATP.

21. Belkhadir Y, Subramaniam R, Dangl JL: Plant disease resistanceprotein signaling: NBS-LRR proteins and their partners.Curr Opin Plant Biol 2004, 7:391-399.

22. Albrecht M, Domingues FS, Schreiber S, Lengauer T: Structurallocalization of disease-associated sequence variationsin the NACHT and LRR domains of PYPAF1 and NOD2.FEBS Lett 2003, 554:520-528.

23. Vetter IR, Wittinghofer A: Nucleoside triphosphate-bindingproteins: different scaffolds to achieve phosphoryl transfer. QRev Biophys 1999, 32:1-56.

24. Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW:Genome-wide analysis of NBS-LRR-encoding genes inArabidopsis. Plant Cell 2003, 15:809-834.

25. Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S,Sobral BW, Young ND: Plant disease resistance genesencode members of an ancient and diverse protein familywithin the nucleotide-binding superfamily. Plant J 1999,20:317-332.

26. Tameling WIL, Elzinga SD, Darmin PS, Vossen JH, Takken FLW,Haring MA, Cornelissen BJC: The tomato R gene products I-2and Mi-1 are functional ATP binding proteins with ATPaseactivity. Plant Cell 2002, 14:2929-2939.

27. Hanson PI, Whiteheart SW: AAA+ proteins: have engine, willwork. Nat Rev Mol Cell Biol 2005, 6:519-529.

28.��

Kim HE, Du F, Fang M, Wang X: Formation of apoptosome isinitiated by cytochrome c-induced dATP hydrolysis andsubsequent nucleotide exchange on Apaf-1. Proc Natl Acad SciUSA 2005, 102:17545-17550.

This paper solves a long dispute about whether APAF-1 requires ATPhydrolysis for function. The authors elegantly show that, in its restingstate, APAF-1 is bound to (d)ATP. Upon activation of the protein bycytochrome c binding, one single round of hydrolysis occurs, resulting inan intermediate ADP-bound state of the protein. In the absence of (d)ATP,the protein forms an inactive high molecular weight aggregate, whereas inthe presence of this nucleotide, the bound (d)ADP is exchanged for(d)ATP, initiating assembly of the apoptosome.

29.��

Tameling WIL, Vossen JH, Albrecht M, Lengauer T, Berden JA,Haring MA, Cornelissen BJC, Takken FLW: Mutations in the



NB-ARC domain of I-2 that impair ATP hydrolysis causeautoactivation. Plant Physiol 2006, 140:1233-1245.

The authors describe the first example of an NBS-LRR protein in whichnucleotide binding and hydrolysis is coupled to a biological function.Biochemical analysis of two autoactivating mutants of the tomato I-2protein reveals that these mutants are affected in ATP hydrolysis but notnucleotide binding. Furthermore, the authors provide evidence that theADP- and ATP-bound states have a different stability, indicating differentprotein conformations. On the basis of these observations, the authorspropose a model to describe how NBS-LRR activation is regulated by theNB-ARC domain.

30. Yu X, Acehan D, Menetret JF, Booth CR, Ludtke SJ, Riedl SJ,Shi Y, Wang X, Akey CW: A structure of the human apoptosomeat 12.8 A resolution provides insights into this cell deathplatform. Structure 2005, 13:1725-1735.

31. Iyer LM, Leipe DD, Koonin EV, Aravind L: Evolutionary historyand higher order classification of AAA+ ATPases. J Struct Biol2004, 146:11-31.

32. Ogura T, Wilkinson AJ: AAA+ superfamily ATPases: commonstructure – diverse function. Genes Cells 2001, 6:575-597.

33. Moffett P, Farnham G, Peart J, Baulcombe DC: Interactionbetween domains of a plant NBS-LRR protein indisease resistance-related cell death. EMBO J 2002,21:4511-4519.

34.��

Mestre P, Baulcombe DC: Elicitor-mediated oligomerization ofthe tobacco N disease resistance protein. Plant Cell 2006,18:491-501.

This elegant study shows, for the first time, the oligomerisation of an NBS-LRR protein upon application of the matching Avr. By silencing knowndownstream signalling components, the authors show that oligomerisa-tion is a very early event in defence signalling. Furthermore, pull-downexperiments using separate domains are used to demonstrate that theTIR domains show a weak homomeric interaction that is independent ofthe Avr. For the full-length protein to oligomerise, both the Avr and an

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intact P-loop in the NB-ARC are required, but a loss-of-function mutationin the RNBS-A domain does not affect the capability to oligomerise.

35. Hwang CF, Williamson VM: Leucine-rich repeat-mediatedintramolecular interactions in nematode recognition and celldeath signaling by the tomato resistance protein Mi.Plant J 2003, 34:585-593.

36. Bendahmane A, Farnham G, Moffett P, Baulcombe DC:Constitutive gain-of-function mutants in a nucleotide bindingsite-leucine rich repeat protein encoded at the Rx locus ofpotato. Plant J 2002, 32:195-204.

37. Rathjen JP, Moffett P: Early signal transduction events inspecific plant disease resistance. Curr Opin Plant Biol 2003,6:300-306.

38. Tian D, Traw MB, Chen JQ, Kreitman M, Bergelson J: Fitnesscosts of R-gene-mediated resistance in Arabidopsis thaliana.Nature 2003, 423:74-77.

39. Nimchuk Z, Eulgem T, Holt BF III, Dangl JL: Recognition andresponse in the plant immune system. Annu Rev Genet 2003,37:579-609.

40. Bieri S, Mauch S, Shen QH, Peart J, Devoto A, Casais C, Ceron F,Schulze S, Steinbiss HH, Shirasu K et al.: RAR1 positivelycontrols steady state levels of barley MLA resistance proteinsand enables sufficient MLA6 accumulation for effectiveresistance. Plant Cell 2004, 16:3480-3495.

41. Yu X, Wang L, Acehan D, Wang X, Akey CW: Three-dimensionalstructure of a double apoptosome formed by the DrosophilaApaf-1 related killer. J Mol Biol 2006, 355:577-589.

42. Aravind L, Iyer LM, Leipe DD, Koonin EV: A novel family of P-loopNTPases with an unusual phyletic distribution andtransmembrane segments inserted within the NTPasedomain. Genome Biol 2004, 5:R30.

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Resistance proteins: molecular switches of plant defence