Molecular Plant Advance Access originally published online on March 21, 2008
Molecular Plant 2008 1(3):401-410; doi:10.1093/mp/ssn009
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Transcomplementation, but not Physical Association of the CC-NB-ARC and LRR Domains of Tomato R Protein Mi-1.2 is Altered by Mutations in the ARC2 Subdomain
a Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, PO Box 94062, 1090 GB Amsterdam, the Netherlands
b Max Planck Institute for Informatics, Stuhlsatzenhausweg 85, 66123 Saarbrücken, Germany
1 To whom correspondence should be addressed. E-mail takken{at}science.uva.nl, fax +31 205257934, tel. +31 205257795.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Race-specific disease resistance in plants is mediated by Resistance (R) proteins that recognize pathogen attack and initiate defence responses. Most R proteins contain a central NB-ARC domain and a C-terminal leucine-rich repeat (LRR) domain. We analyzed the intramolecular interaction of the LRR domain of tomato R protein Mi-1.2 with its N-terminus. We expressed the CC-NB-ARC and LRR parts in trans and analyzed functional transcomplementation and physical interactions. We show that these domains functionally transcomplement when expressed in trans. Known autoactivating LRR domain swaps were found to induce a hypersensitive response (HR) upon co-expression. Likewise, autoactivating mutants in the NB subdomain transcomplemented to induce HR. Point mutations in the ARC2 subdomain that induce strong autoactivation in the full-length Mi-1.2 protein, however, fail to induce HR in the transcomplementation assay. These data indicate distinct functions for the NB-ARC subdomains in induction of HR signalling. Furthermore, dissociation of the LRR is not required to release its negative regulation, as in all combinations of CC-NB-ARC and LRR domains tested, a physical interaction was observed.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Race-specific resistance in plants is mediated by specific surveillance proteins called resistance (R) proteins (Tameling and Takken, 2007). Upon pathogen perception, R proteins trigger a strong defence response, often culminating in a hypersensitive response (HR)—a form of programmed cell death mechanism aimed at restricting pathogen proliferation. Most R proteins are large intracellular proteins that contain a central Nucleotide Binding (NB-) domain fused to a C-terminal leucine-rich repeat (LRR) domain.
The LRR domain consists of a variable number of leucine-rich repeat units and harbours negative as well as positive regulatory functions. The positive regulatory role for R protein function is apparent as the LRR domain is indispensable and deletion normally does not result in constitutive activity (Bendahmane et al., 2002; Hwang and Williamson, 2003; Rathjen and Moffett, 2003). The negative regulatory function becomes clear when an LRR is improperly matched to the rest of an R protein. For instance, swapping the Mi-1.2 LRR region into paralog Mi-1.1 results in autoactivity, namely activation of defence responses in the absence of the pathogen. The reciprocal swap results in loss of nematode resistance in transgenic roots (Hwang et al., 2000). Likewise, autoactivation upon LRR exchange has been observed for maize Rp1 homologues (Sun et al., 2001) and by swapping parts of the potato Rx LRR domain for the corresponding (highly homologous) Gpa2 sequences (Rairdan and Moffett, 2006). These experiments suggest that the LRR domain interacts with the rest of the protein and that improper pairing can result in de-repression of the protein. Direct physical and functional interaction of the R protein LRR domain with the rest of the protein has indeed been shown, first for potato Rx (Moffett et al., 2002) and later for tobacco N (Ueda et al., 2006), pepper Bs2 (Leister et al., 2005) and Arabidopsis RPS5 (Ade et al., 2007). Altogether, these observations indicate that subtle intramolecular interactions between the LRR domain and the N-terminus are required for proper R protein function.
The most well conserved protein domain in NB-LRR proteins is the NB-ARC domain. This nucleotide-binding domain is found in proteins such as human Apaf-1, plant R proteins and C. elegans Ced-4 (ARC), hence its name, NB-ARC. When the crystal structures of NB-ARC domains were established for Apaf-1 and CED-4 (Riedl et al., 2005; Yan et al., 2005), it became apparent that the NB-ARC consists of structurally defined subdomains. No plant NB-ARC domain has been crystallized so far, but, based on homology modeling, it has been proposed that the R protein NB-ARC consists of three subdomains: a nucleotide-binding fold (NB), a four-helix bundle (ARC1) and a winged-helix fold (ARC2) (Albrecht and Takken, 2006). These subdomains are spatially well defined and are proposed to have functionally distinct roles, as elaborated below. The NB-ARC domain of R proteins I-2, Mi-1 and N has been shown to be a functional module for nucleotide binding and hydrolysis (Tameling et al., 2002; Ueda et al., 2006). For I-2, it has been proposed that the ATP-bound state represents the active state and hydrolysis of the nucleotide is proposed to return the R protein into its resting state (Takken et al., 2006; Tameling et al., 2006). The nucleotide is likely bound at the interface between the NB, the ARC1 and ARC2, (Takken et al., 2006).
Some aspects of the ARC1 subdomain function have been revealed by a series of domain swaps between R proteins Rx and Gpa2 (Rairdan and Moffett, 2006). These studies showed that the Rx ARC1 subdomain is necessary for the interaction of the N-terminus with the LRR domain. LRR binding seems to be a general feature of the ARC1 subdomain, since the Rx N-terminus was able to interact with the LRR domains of several other NB-LRR proteins. An extensive interaction surface with several individual contacts quantitatively contributing to the interaction was shown between the ARC1 and the LRR domain (Rairdan and Moffett, 2006). Together with the fact that no autoactive mutants have been described in this region, this suggests that the ARC1 subdomain is merely a scaffold maintaining the intramolecular interactions with the LRR domain.
The ARC2 subdomain is proposed to relay pathogen recognition, mediated by the LRR domain into changes in R protein conformation, unleashing its downstream signaling potential. Swapping the Gpa2 ARC2 subdomain with the Rx ARC2 domain results in a constitutively active chimera (Rairdan and Moffett, 2006). Apparently, the ARC2 subdomain of Gpa2 and the LRR domain of Rx are sufficiently compatible to induce HR, but are not harmonized to prevent autoactivation. A role for the ARC2 subdomain in signal transduction fits with the fact that many autoactivating mutations in R proteins map to the ARC2 (Bendahmane et al., 2002; Shirano et al., 2002; de la Fuente van Bentem et al., 2005; Howles et al., 2005; Gabriëls et al., 2007). Support for such a regulatory function for the ARC2 is provided by mutational analysis of the conserved MHD motif in the ARC2 and modeling of the I-2 NB-ARC domain on the related human protein Apaf-1 (van Ooijen et al., 2008).
NB-LRR proteins can be roughly divided into two main subclasses, depending on their N-terminal domain flanking the NB-ARC (Tameling and Takken, 2007; van Ooijen et al., 2007). This domain is either a region with homology to the Drosophila Toll and human Interleukin-1 Receptor proteins (the TIR domain), or a region of 120–200 amino acids without such homology, sometimes containing coiled-coil (CC) motifs. In solanaceous plants, several CC-NB-ARC-LRR proteins have a significantly larger N-terminal domain, and the presence of this extended N-terminus might indicate a difference in function between these and the ‘regular’ CC-NB-ARC-LRR proteins (Mucyn et al., 2006; Rairdan and Moffett, 2007). The R protein Mi-1.2 (Milligan et al., 1998; Vos et al., 1998) belongs to the group of CC-NB-ARC-LRR proteins with an extended N-terminus. Mi-1.2 is activated by attempted host cell manipulation by three different root-knot nematode species (Meloidogyne spec.). In later studies, Mi-1.2 was also found to mediate resistance against infestations of ploem-feeding white-fly and aphid pests (Martinez de Ilarduya et al., 2003; Nombela et al., 2003; Li et al., 2006). Mi-1.2 is a member of a small gene family and is located in a cluster containing a paralog (Mi-1.1) on chromosome 6. Mi-1.1 shares 91% amino-acid identity with Mi-1.2 (Milligan et al., 1998) and, like Mi-1.2, is transcribed, although no known resistance specificity has been assigned to Mi-1.1. Numerous domain swaps have been made between Mi-1.1 and Mi-1.2 to generate data on regions of the gene that are important for negative and positive regulation (Hwang et al., 2000; Hwang and Williamson, 2003).
Here, we address various aspects of the function of the Mi-1.2 NB-ARC subdomains in relation to its interaction with the LRR domain. Autoactivating mutants in the NB subdomain were found to trigger HR upon co-expression of the separate CC-NB-ARC and LRR parts. In contrast, autoactivating mutations in the ARC2 subdomain fail to transcomplement and induce HR. These observations suggest that the ability to transcomplement depends on the NB-ARC subdomain affected by the mutation. In all combinations of CC-NB-ARC and LRR domains analyzed, the physical interactions were not abolished, indicating that no gross changes in intramolecular interactions are associated with the induction of HR signalling. These results confirm a model suggesting a distinct functional role for each of the three NB-ARC subdomains. The NB subdomain is important for nucleotide binding and for the enzymatic activity of the NB-ARC domain, the ARC1 has a general LRR interacting role, and the ARC2 subdomain plays a dual regulatory role in sensing the nucleotide and translating pathogen perception into signalling.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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CC-NB-ARC and LRR Expressed in trans Complement to Reconstitute a Signalling-Competent Molecule
A chimaeric protein that consists of the CC-NB-ARC part of Mi-1.1 and the LRR domain of Mi-1.2 (referred to as Mi-DS4) induces a hypersensitive response (HR) upon transient expression in Nicotiana benthamiana leaves when expressed from the endogenous Mi-1.1 promoter (Hwang et al., 2000). Here, we show that expression of this chimera Mi-DS4, but not Mi-1.1 or Mi-1.2, driven from the strong CaMV 35S promoter induces a strong necrosis 2 d after infiltration (Figure 1, left panel). This cell death is clearly visualized upon trypan blue staining of this leaf, to stain dead cells blue. Expression of Mi-1.1 or Mi-1.2 proteins induces a background level of cell death visualized by a light-blue hue upon trypan blue staining (Figure 1, right panel).
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To investigate the molecular basis behind the autoactive incompatibility between the Mi 1.1 CC-NB-ARC and Mi 1.2 LRR domains in Mi-DS4, and not, for example, in the reciprocal domain swap (referred to as Mi-DS2), functional complementation was analyzed when the CC-NB-ARC and LRR domains were expressed in trans. To this end, the corresponding Mi-1.1 and Mi-1.2 parts were placed behind the 35S promoter and the four combinations were expressed in overlapping circles on N. benthamiana leaves by agroinfiltration. In this way, the effect of expression of the separate domains and combinations of two can be compared on a single leaf. In the infiltrated leafs depicted in Figure 2, the NB-ARC domains are expressed in the upper circles whereas the LRRs are expressed in the lower circles.
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Similar to the full-length proteins (Figure 1), HR signalling was initiated upon co-expression of Mi-1.1 CC-NB-ARC and Mi-1.2 LRR (overlapping region Figure 2B). To visualize the cell death, the same leaves were stained with trypan blue (right panels in Figure 2). In contrast to Mi-DS4, the reconstituted Mi-1.1 (Figure 2A), Mi-DS2 (Figure 2C) or Mi-1.2 (Figure 2D) did not trigger HR. HR was also not observed in the non-overlapping regions, showing that expression of the separate CC-NB-ARC or LRR domain induces only a basal level of cell death. To be able to detect expression of the protein domains in planta, the Mi-DS4 constituents were fused to various tags at either their N- or C-terminus and analyzed for functional transcomplementation. Of all combinations tested, only co-expression of non-tagged CC-NB-ARC protein and either non-tagged or N-terminally TAP-tagged LRR protein triggered HR. As the latter combination is still functional, we used it for all our transcomplementation assays (including Figure 2).
These data show that we can functionally reconstitute an autoactivating Mi-1 chimaeric swap protein able to signal HR by expressing the Mi-1.1 CC-NB-ARC and Mi-1.2 LRR domains in trans. Because expression of the separate domains does not induce an HR, these data suggest that the two domains interact to activate defence signalling. The combination provides all positive regulatory functions required to induce an HR, but lacks the negative regulation required to suppress this signalling in the absence of a pathogen.
Autoactivation in Mi-DS4 Does Not Require Dissociation of the LRR Domain from the N-terminus
To analyze whether physical intramolecular interactions are responsible for the autoactivation phenotype of Mi-DS4, combinations of CC-NB-ARC and TAP-LRR constructs of Mi-1.1 and Mi-1.2 were co-expressed. On a Western blot of total protein lysates of the agroinfiltrated leaves, a band migrating at the expected size for the CC-NB-ARC protein (
100 kDa) is visualized (Figure 3, upper panel) using an Mi-1 antibody raised against the N-terminal region of Mi-1 not containing the LRR domain. The antibody also recognizes the TAP-LRR fusion protein (63 kDa) because of the general IgG-binding properties of the ProtA domain in the TAP tag.
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Mi antibody. The upper panel represents an immunoblot of total protein extracts (input); the lower panel represents the TAP-precipitated protein complex after IP.To test physical interactions between the two protein domains, the TAP-LRR fusion protein was immunoprecipitated using agarose beads loaded with human IgG. Human IgGs were used to avoid cross-reactivity with the anti-rabbit secondary antibody needed for detection of the Mi-1 protein in subsequent immunoblots. The precipitated protein-complex was proteolytically cleaved from the beads using the specific TEV protease site in the TAP tag (Rohila et al., 2004).
After pull-down, co-purification of CC-NB-ARC protein was analyzed using the Mi-1 antibody (Figure 3, lower panel). Co-purification was found with all four combinations of Mi-1.1 and Mi-1.2 domains. Apparently, the interaction of LRR domains with CC-NB-ARC is not restricted to their endogenous partners.
An interesting observation is co-precipitation of a band corresponding to the size of the TAP-LRR protein (63 kDa). As mentioned above, the Mi antibody does not recognize the cleaved non-tagged LRR domain and the ProtA epitope should remain bound to the beads. This observation suggests either that more than one LRR domain is present in one complex or that the conditions of the TEV protease cleavage are facilitating non-enzymatic dissociation of the complex from the beads. Since this phenomenon is not observed with other TAP-tagged proteins (data not shown), the latter hypothesis is unlikely. In any case, co-precipitation of CC-NB-ARC protein in the presence, and not in the absence, of TAP-LRR protein shows that both are present in one complex and the interaction is specific.
This experiment clearly shows that a physical interaction is present in the transcomplementing Mi-DS4 combination. Based on the intensity of the bands the stoichiometry of the interaction is similar to that of the other combinations, implying that autoactivation does not require gross changes (i.e. dissociation) in intramolecular interactions between the N-terminus and the LRR domain.
Identification of Autoactivating Mutations in the NB-ARC Domain
The Mi-1.1 and Mi-1.2 proteins are 91% identical and the differences are scattered throughout the protein, making it difficult to pinpoint the residues responsible for autoactivating incompatibilities. To be able to distinguish between positive and negative regulatory functions in the CC-NB-ARC and LRR combination, we focused on specific point mutations in the NB-ARC domain of Mi-1.2 that lead to autoactivation: T557S, H840A and D841V (Gabriëls et al., 2007; van Ooijen et al., 2008). Autoactivating mutants by definition represent HR signalling-competent molecules, in contrast to loss-of-function mutants that can be affected in various aspects of R protein function.
Autoactivating mutations in the NB-ARC domain map to the NB or to the ARC2 subdomain. The position of these mutations is visualized in a 3D model of the Mi-1.2 NB-ARC domain (Figure 4). This model is based on homology modeling of the Mi-1.2 NB-ARC on the crystal structure of the ADP-bound state of the NB-ARC domain of the human protein Apaf-1 (Riedl et al., 2005).
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Based on this model, we selected two additional mutants. One of these represents a mutant in the NB subdomain (D630E) that maps to the Walker B motif (Figure 4). The corresponding residue has also been shown to lead to autoactivation in I-2 (Tameling et al., 2006). The second mutant is a proposed loss-of-function mutant. In this double mutant (KT556/557AA), two highly conserved amino acids in the nucleotide-binding P-loop are changed to alanine, thereby presumably abolishing the ability to bind nucleotides (Figure 4).
As can be clearly seen in the 3D model, all mutations used in this study cluster at the nucleotide-binding interface, showing that, although the mutations map to different regions in a linear sequence, they cluster spatially.
Mutations in the ARC2, but not in the NB Subdomain, Abolish Transcomplementation
To compare the extent of cell death mediated by the five Mi-1.2 NB-ARC mutants, they were transiently expressed in N. benthamiana leaves using agroinfiltration. Figure 5 shows the infiltrated leafs 2 d after infiltration. At this time-point, the ARC2 mutants H840A (bottom left) and D841V (top right) induced a clear HR, whereas the NB mutants T557S (bottom right) and D630E (middle right) do not yet induce a specific cell death response. The T557S-initiated HR becomes apparent after 3 d (Gabriëls et al., 2007), whereas the D630E mutation leads to visual necrosis 5 d after infiltration (data not shown). Of the four autoactivating mutants, H840A and D841V are the fastest, whereas the two NB mutants (T557S and D630E) induce a relatively slow HR. No HR was observed with wild-type Mi-1.2 or with the KT556/557AA mutant.
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To test whether transcomplementation of CC-NB-ARC and LRR domains is affected in any of these NB-ARC mutants, we co-expressed wild-type and the five mutant CC-NB-ARC proteins and wild-type TAP-LRR protein in overlapping circles in N. benthamiana leaves. In all leaves pictured in Figure 6, the NB-ARC domains are infiltrated in the upper circle and the lower circle represent the region of Mi-1.2 TAP-LRR expression. As expected, the wild-type (Figure 6A) and predicted loss-of-function KT556/557AA (Figure 6B) NB-ARC domains did not induce HR when co-expressed with the LRR domain. The absence of HR was verified by trypan blue staining of the same leaves (right panels).
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The NB mutants T557S and D630E did transcomplement and reconstituted autoactivity (overlapping zone in Figure 6C and 6D). Similarly to the full-length protein, the D630E mutant induced a milder response than the T557S mutant, but HR was clearly visible in the overlapping region upon trypan blue staining (Figure 6D, right panel). These results show that NB subdomain mutants are able to induce HR when the LRR domain is expressed in trans. The cell death response is faster than that induced by the full-length protein, presumably due to the higher expression levels observed for the separate domains (data not shown).
In contrast to the NB mutants, co-expression of CC-NB-ARC variants containing the strong ARC2 autoactivation mutants H840A or D841V reproducibly fails to induce HR responses (Figure 6E and 6F). Apparently, the mutations that result in the strongest autoactivation phenotypes in cis (Figure 5) cannot complement with the LRR domain to reconstitute a functional molecule when expressed in trans (Figure 6). These results show that functional transcomplementation of autoactivity depends on the subdomain of the NB-ARC that is affected.
Mutations in the NB-ARC Domain Do Not Lead to Dissociation of the LRR Domain
To investigate whether the observed differences in the transcomplementation assay can be explained by a change in physical interaction of CC-NB-ARC and LRR, combinations of mutant CC-NB-ARC and TAP-LRR proteins were co-expressed. The Western blot of the total protein lysates (Figure 7, upper panel) shows equal expression of the CC-NB-ARC variants and of the Mi-1.2 TAP-LRR proteins. Both migrate according to their predicted masses of 100 and 63 kDa.
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The Mi-1 protein-complex was precipitated using human IgG-beads and released using TEV protease. Co-precipitation of CC-NB-ARC domains with TAP-LRR was analyzed on Western blots using the Mi-1 antibody. As can be seen in the lower panel of Figure 7, all CC-NB-ARC variants tested interact equally well with the Mi-1.2 LRR domain. These interactions show that even though transcomplementation of the CC-NB-ARC and LRR domains depends on the location of mutations in the NB and ARC2 subdomains, there are no detectable changes in intramolecular interactions in these mutants. Apparently, interaction of the LRR domain with its N-terminus does not depend on the functionality of the combination, and is not sufficient for autoactivation.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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We have addressed the molecular background of autoactivating domain swaps of the Mi-1.2 and Mi-1.1 proteins, and of autoactivating and loss-of-function point mutations in the NB-ARC domain of Mi-1.2, summarized in Table 1. Using an Mi-1 antibody and a tagged LRR construct, we have been able to study intramolecular interaction of the LRR domain and the CC-NB-ARC part of Mi-1 paralogs and NB-ARC mutants. The set of mutants depicted in the model in Figure 4 enabled us to investigate the effect of mutations in NB-ARC subdomains on transcomplementation and intramolecular interaction with the LRR domain. In all combinations of Mi-1 LRR and CC-NB-ARC variants, a physical interaction was observed. Apparently, autoactivation and loss-of-function mutations in NB and ARC2 studied here do not result in dissociation of the CC-NB-ARC and LRR domains. The ability to transcomplement, however, was found to be dependent on the NB-ARC subdomain affected by the autoactivating mutation.
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The observed constitutive interaction between the CC-NB-ARC and LRR domain appears to contrasts with a previously proposed model based on genetic data and domain swap analyses (e.g. for Mi-DS4) that suggest dissociation of these domains upon (auto)activation (Hwang et al., 2000; Hwang and Williamson, 2003). However, it is likely that the functional interaction between these two domains is lost, even though the physical interactions remain. The observed interactions of the LRR domain with the CC-NB-ARC parts in Mi-DS4, Mi-DS2, and both wild-type proteins show that association of the LRR domain with the N-terminus is not sufficient to induce HR, nor to repress it. Since Mi-1.1 and Mi-DS2 failed to confer nematode resistance (Hwang et al., 2000), the combination of the genuine CC-NB-ARC and LRR (Mi-1.2) domains is required to perceive the nematode-derived stimulus and trigger defence.
To explain how the point mutations in the NB-ARC affect the activation state, the NB-ARC domain of Mi-1.2 was modeled on the APAF-1 template (Figure 4). The replacement of the highly conserved lysine (K556) and threonine (T557) in the P-loop of the NB subdomain to alanine is predicted to disrupt nucleotide binding. In the structure of Apaf-1, the P-loop provides seven of the eight direct hydrogen bonds to the bound ADP (the eighth is provided by the histidine in the MHD motif) (Riedl et al., 2005), showing the importance of this motif for nucleotide binding. Direct support for a role in nucleotide binding by the P-loop in R proteins is provided by the observation that replacement of the corresponding lysine in I-2 results in a nucleotide-binding mutant with a loss-of-function phenotype (Tameling et al., 2002). The T557S mutation in the P-loop of Mi-1.2 apparently does not abolish nucleotide binding, since this mutant has an autoactivation phenotype. Possibly, the orientation of the bound nucleotide is aberrant in this mutant and mimics the activated state, or, alternatively, the mutation reduces the ATPase activity. The latter hypothesis is supported by analysis of two autoactivation mutations in the NB subdomain of I-2 that were shown to have a reduced hydrolysis capacity (Tameling et al., 2006). One of them is the D283E mutation in the Walker B motif. This acidic residue is thought to be a catalytic base that is required for ATP hydrolysis by P-loop ATPases (Leipe et al., 2004). Based on the structural conservation, we therefore propose the same role for the analogous D630E mutation in Mi-1.2 and predict that it will be a hydrolysis mutant.
The autoactivating phenotypes observed upon mutation of the MHD motif in the ARC2 are likely based on a destabilized interaction between this domain and the nucleotide. A CC-NB-ARC domain harbouring an autoactivating mutation in the MHD motif is not able to induce an HR by itself (Figures 2 and 6), but requires the LRR domain in cis (Figure 5). When these domains are expressed in trans, HR signalling is not induced, although their interaction is not compromised. Apparently, the positive regulatory function of the LRR on the CC-NB-ARC domain, required for the protein to activate defence signalling, is lost when presented in trans. We can exclude that the separated LRR domain exhibits dominant negative regulation of the MHD mutants, since co-expression of the LRR with full-length MHD mutants does not suppress HR (data not shown). These observations suggest a sensory and regulatory role for the ARC2 subdomain in mediating LRR-derived signals.
In Rx, the corresponding MHD mutant D460V can still transcomplement with the LRR domain (Moffett et al., 2002). A possible basis for this difference is that Rx belongs to a different group of CC-NB-ARC-LRR proteins. It would be interesting to examine whether the ability to transcomplement the MHD mutant phenotype is NB-LRR (sub)class-specific.
To summarize, this study provides the first data on intramolecular interaction between domains of an NB-LRR R protein with an extended CC domain. The robust interaction between LRR and CC-NB-ARC domains supports the hypothesis by Rairdan and Moffett (2006) that the responsible interface consists of several contact points and can therefore not readily be disturbed. Our results support a distinct role for the three NB-ARC subdomains. The NB subdomain is involved in nucleotide binding and hydrolysis, and mutations that disrupt binding result in a loss-of-function phenotype whereas putative hydrolysis mutants are autoactivating (Figure 5). Autoactivity conferred by NB subdomain mutants is presumably caused by a shift in the equilibrium towards the ATP-bound state, which is proposed to be the active state (Tameling et al., 2006). In the NB subdomain mutants, the negative regulation exerted by the LRR is lost, but the positive regulatory functions are required and sufficient to confer the autoactivation phenotype (Figure 6). In contrast, mutation of the ARC2 also alleviates negative regulation, but the positive regulation of the LRR domain can only be provided in cis (Figure 5) and not in trans (Figure 6), suggesting that the interaction between these domains is altered, even though interaction is not lost (Figure 7). Binding to the LRR is therefore likely to be mediated by the ARC1 subdomain, in accordance with the observations described for Rx (Rairdan and Moffett, 2006).
Altogether, our data support a model in which perception of a pathogen-derived factor by the LRR domain results in an alteration of the interaction interface between the LRR domain and the ARC2 subdomain, resulting in a release of the negative regulation exerted by the LRR on the NB-ARC domain and transition to the activated state.
Further studies will be required to understand how positive and negative regulation by the LRR on the NB-ARC domain is executed. However, the analysis of intramolecular interactions aid to reveal parts of the basic mechanisms underlying activation. To fully understand the molecular dynamics of the interactions, crystal structures of full-length R proteins in the ATP- and ADP-bound state will be essential. Changes in conformation might be subtle and, rather than complete association or dissociation of (sub)domains, intricate changes in binding interfaces might be sufficient to unleash the signalling potential of an R protein.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Construction of Binary Vectors
All PCR primers used in this study (marked FPxxx) were purchased from MWG (Germany), and are listed in Table 2. Construction of a binary vector expressing Mi-1.2 (pG74) was described elsewhere (Gabriëls et al., 2007). Mi-1.1 was amplified from plasmid G3 (Keygene N.V.) with primers FP764 and FP766. Gateway attB flanks were added by adapter PCR using primers FP872 and FP873. The PCR product was transferred via entry clone pDONR207 (Invitrogen) to binary vector CTAPi (Rohila et al., 2004) by the Gateway one-tube protocol for cloning attB-PCR products directly into destination vectors (Invitrogen). In the same way, Mi-DS4 was amplified from plasmid Mi-DS4 (Hwang et al., 2000) and cloned into CTAPi. Note that the endogenous stop codon remains present in these constructs, so there is no translational fusion to the TAP tag.
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Constructs expressing the Mi-1.2 mutants T557S, H840A, and D841V have been described elsewhere (Gabriëls et al., 2007; van Ooijen et al., 2008). D630E was generated by overlap extension PCR using primer sets FP554/FP212 and FP182/FP555 on template pSE23 (Gabriëls et al., 2007). The 1330 and 260-bp pcr products were joined in a second amplification step using FP182/FP212. A 325-bp restriction fragment generated with AgeI and XhoI containing the desired mutation was used to replace the wild-type sequence in pSE23 to obtain pG23. The full-length Mi-1.2 D630E sequence was amplified using FP764 and FP766, Gateway attB sites were added with FP872 and FP873 and the resulting fragment was transferred to binary vector CTAPi, as described above. The Mi-1.2 KT556/557AA construct was generated by circular mutagenesis (Hemsley et al., 1989) on template pG104 (van Ooijen et al., 2008) using FP1099/FP1102 to obtain pG108. An Eco72I/Bsp119I restriction fragment carrying the mutation was exchanged with that in pG74 to obtain Mi-1.2 KT556/557AA.
Mi-1.1 and Mi-1.2 LRR sequences (residues 1–899) were amplified from G3 and pKG6210 (Keygene N.V.), respectively, using primers FP857 and FP766. Gateway attB flanks were added using FP872 and FP873 and the fragment was transferred to pDONR207 by a Gateway BP reaction (Invitrogen) and subsequently to NTAPi (Rohila et al., 2004) to obtain N-terminally TAP-tagged LRR domains.
All CC-NB-ARC (residues 900–1258) constructs were generated by the Gateway one-tube protocol for cloning attB fragments directly in destination vectors (Invitrogen). Primers FP764 and FP859 were used on PCR templates pKG6210 (Mi-1.2, Keygene N.V.) and G3 (Mi-1.1, Keygene N.V.), or the full-length clones of the Mi-1.2 mutants described above (and in Gabriëls et al., 2007; van Ooijen et al., 2008). Note that a stop codon has been incorporated in the reverse primer to prevent translational fusion to the TAP tag in these CC-NB-ARC constructs.
Agrobacterium-Mediated Transient Transformation
A. tumefaciens strain GV3101(pMP90) (Koncz and Schell, 1986) was transformed with binary constructs (Takken et al., 2000) and grown to OD600 = 0.8 in LB-Mannitol (10 g l–1 tryptone, 5 g l–1 yeast extract, 2.5 g l–1 NaCl, 10 g l–1 mannitol) medium supplemented with 20 µM acetosyringone and 10 mM MES pH 5.6. Cells were pelleted and resuspended in infiltration medium (1 MS salts, 10 mM MES pH 5.6, 2% w/v sucrose, 200 µM acetosyringone) and infiltrated at OD600 = 0.2 (for LRR constructs) or 1 (for full-length or CC-NB-ARC constructs) into 4–5-week-old N. benthamiana leaves.
Trypan Blue Staining
Leaves were boiled for 5 min in a 1:1 mixture of ethanol and 0.33 mg ml–1 trypan blue in lactophenol, and destained overnight in 2.5 g ml–1 chloral hydrate in H2O.
Protein Extraction, Immunoprecipitation and Western Blotting
Infiltrated leaves were harvested and pooled 24 h after agroinfiltration and frozen in liquid nitrogen. After grinding the tissue, it was allowed to thaw in 2 ml protein extraction buffer per gram of tissue (25 mM Tris pH 8, 1 mM EDTA, 150 mM NaCl, 5 mM DTT, 0.1% NP-40, 1 Roche Complete protease inhibitor cocktail and 2% Poly-(Vinyl-Poly-Pyrolidone)). Extracts were centrifuged at 12 000 g at 4°C for 10 min and the supernatant was passed over four layers of Miracloth to obtain a total protein lysate. 1 ml of total protein lysate was incubated with 20 µl human IgG-agarose beads (Sigma) rotating head-over-head at 4°C for 1 h. Beads with bound protein complexes were pelleted at 2000 rpm for 10 s and washed four times with 1 ml ice-cold TEV cleavage buffer (10 mM Tris pH 8, 150 mM NaCl, 0.5 mM EDTA, 0.1% NP-40 and 1 mM DTT). 100 µl TEV cleavage buffer and 10 units recombinant AcTEV protease (Invitrogen) were added to the pelleted beads and the proteolytic reaction was carried out shaking vigorously for 1.5 h at 16°C. The supernatant was mixed with Laemmli sample buffer, run on 8% SDS-PAGE gels and blotted on PVDF membranes using semi-dry blotting. 5% skimmed milk powder was used as blocking agent. As a primary antibody, a 1:4000 dilution of the Mi-1 antibody (van Ooijen et al., 2008) was used and, as a secondary antibody, a 1:4000 dilution of goat-anti-rabbit linked to either horseradish peroxidase (Figure 3) or alkaline phosphatase (Figure 7). The signal was visualized by measuring either luminescence (ECL and BioMax MR film (Kodak) (Figure 3) or fluorescence (ECF and detection by phospho-imager (STORM)) (Figure 7).
3D Structure Model of the Mi-1.2 NB-ARC Domain
A pairwise sequence-structure alignment of tomato R protein Mi-1.2 and human Apaf-1 was constructed based on the multiple alignment of R protein NB-ARC domains described in (van Ooijen et al., 2008). The 3D-modeling server WHAT IF (Vriend, 1990) returned a full-atom structure model of the NB-ARC domain of Mi-1.2. The structure of Apaf-1 (PDB code 1z6t, chain A) comprises the residues 108–450 (UniProt sequence O14727
[GenBank]
) and is mapped onto Mi-1.2 residues 505–852 (UniProt sequence O81137). The protein structure image of the model was illustrated using PyMOL (www.pymol.org).
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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G.v.O. is supported by the Research Council for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific. Research (NWO). G.M. and M.A. have been financially supported by the German National Genome Research Network (NGFN). The research has been conducted in the context of the BioSapiens Network of Excellence funded by the European Commission under grant number LSHG-CT-2003–503265.
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Acknowledgements |
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We would like to thank Harold Lemereis for plant handling. We are grateful to Valerie Williamson for providing us with the Mi-DS4 construct and helpful discussions. No conflict of interest declared.
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