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First published online December 30, 2005; 10.1105/tpc.105.037234 The Plant Cell 18:491-501 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
Elicitor-Mediated Oligomerization of the Tobacco N Disease Resistance Protein[W],[OA]Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom 2 To whom correspondence should be addressed. E-mail david.baulcombe{at}sainsbury-laboratory.ac.uk; fax 44-1603-450011.
Plant nucleotide binding site–leucine-rich repeat (NBS-LRR) proteins are similar to the nucleotide binding oligomerization domain (NOD) protein family in their domain structure. It has been suggested that most NOD proteins rely on ligand-mediated oligomerization for function, and we have tested this possibility with the N protein of tobacco (Nicotiana tabacum). The N gene for resistance to Tobacco mosaic virus (TMV) is a member of the Toll-interleukin receptor (TIR)-NBS-LRR class of plant disease resistance (R) genes that recognizes the helicase domain from the TMV replicase. Using transient expression followed by immunoprecipitation, we show that the N protein oligomerizes in the presence of the elicitor. The oligomerization was not affected by silencing Nicotiana benthamiana ENHANCED DISEASE SUSCEPTIBILITY1 and N REQUIREMENT GENE1 cofactors of N-mediated resistance, but it was abolished by a mutation in the P-loop motif. However, loss-of-function mutations in the RNBS-A motif and in the TIR domain retain the ability to oligomerize. From these results, we conclude that oligomerization is an early event in the N-mediated resistance to TMV.
Plant disease resistance (R) proteins are components of a plant surveillance system that recognize pathogen-derived elicitors and trigger signal transduction cascades, leading to defense. The largest class of plant R proteins contains a nucleotide binding site (NBS) and C-terminal leucine-rich repeats (LRRs) with either a Toll-interleukin receptor domain (TIR) or a loosely defined coiled coil (CC) at their N terminus (Dangl and Jones, 2001  ). These proteins are similar to the mammalian NOD (for nucleotide binding oligomerization domain) protein family, which functions in inflammation and apoptosis (Inohara and Nunez, 2003). R proteins and NOD proteins are alike in their domain structure, in that their N-terminal domains have been implicated in signaling, and in their role in the innate recognition of microorganisms (Inohara and Nunez, 2003).
By analogy with their animal protein homologues, it seemed likely that NBS-LRR proteins would oligomerize in response to pathogen elicitors. It is possible that, like NOD proteins interacting with ligands, they would oligomerize through their NBS domain (Inohara and Nunez, 2003
However, in a study that had the potential to detect elicitor-mediated oligomerization of NBS-LRR R proteins, there was no evidence for homotypic protein–protein interactions. This study involved the CC-NBS-LRR protein Rx that confers resistance to Potato virus X (PVX) upon recognition of the PVX coat protein (CP) (Bendahmane et al., 1999
Here, we describe further investigations of R protein interactions and oligomerization using the tobacco (Nicotiana glutinosa) N protein. N is unlike Rx in that it is a TIR-NBS-LRR rather than a CC-NBS-LRR protein (Whitham et al., 1994
Functional Transient Expression of Epitope-Tagged N Protein The N-mediated response can be observed in tobacco (Nicotiana tabacum) cv Samsun (NN) as a hypersensitive response (HR) after transient expression of the TMV P50 elicitor (Figure 1A). In N-transgenic Nicotiana benthamiana plants (line 310A, carrying N under the control of its native promoter), there is no HR but the N response is manifested as resistance against a green fluorescent protein–tagged version of TMV (TMV:GFP). TMV:GFP induces green fluorescent foci on the inoculated leaves of nontransgenic plants that are not produced on 310A (Figure 1B).
Transiently expressed hemagglutinin (HA)- and myc-tagged versions of the N protein under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator were also functional in the transient expression assay. Thus, transient expression of HA-tagged N genomic sequence in tobacco cv Petite Havana (nn) generated a P50-dependent HR (Figure 1A), and in nontransformed N. benthamiana it suppressed TMV:GFP (Figure 1B). The HR response was not visible when HA-tagged N was expressed in tobacco cv Petite Havana (nn) in the absence of P50 (Figure 1A). The same results were obtained with a myc-tagged version of the protein and with similar constructs coupled to the promoter from N. However, except where stated, the experiments described below were with the 35S promoter constructs. In all instances, the constructs had the 35S rather than the N transcriptional terminator; nevertheless, the encoded wild-type N proteins were functional mediators of TMV resistance. This finding is in contrast with the previous report that the N genomic 3' sequence is required for proper N function (Dinesh-Kumar and Baker, 2000 ). A possible explanation for the difference is our use of a transient assay rather than stable transformation for the expression of N.
N Protein Oligomerizes in Response to Elicitor
Next, we addressed the elicitor-mediated oligomerization of N in different genetic backgrounds in which the N-mediated response was compromised. To do so, we performed our experiments in plants silenced for EDS1, NRG1, and SGT1, genes required for N-mediated resistance (Peart et al., 2002a , 2002b, 2005). Tobacco rattle virus (TRV)-induced silencing of EDS1 and NRG1 had no effect on the P50-dependent interactions of N protein. The N-HA and N-MYC proteins coimmunoprecipitated in extracts of TRV:EDS1- or TRV:NRG1-infected plants, as in plants infected with an empty TRV (TRV:00) (Figure 3A). These results were consistent in three independent experiments, and additional experiments confirmed the predicted loss of TMV resistance in plants infected with the EDS1- and NRG1-silencing constructs (Peart et al., 2002a, 2002b, 2005). Therefore, from these results, we can rule out the possibility that oligomerization is a consequence of the resistance response, and we conclude that elicitor-mediated oligomerization of N protein is upstream of EDS1 and NRG1 in the N-mediated response to TMV.
The level of N protein in the P50-elicited samples was consistently higher than in nonelicited samples (CP) (see Figures 2, 3, 4C, and 5C), suggesting that N is stabilized or solubilized in the presence of the P50 elicitor. However, after silencing of SGT1 with TRV:SGT1, in contrast with the results with TRV:EDS1 and TRV:NRG1, there were lower levels of soluble N and the P50-induced N oligomerization could not be detected (Figure 3A), even after overexposure of the protein gel blot (Figure 3B). The levels of N were so low that we cannot draw any conclusions about the role of SGT1 in N oligomerization. However, these results indicate that SGT1, either directly or indirectly, plays a role in the stabilization of N.
Mutations in Conserved Motifs Affect P50 Elicitor-Mediated Oligomerization and N Protein Stabilization The P-loop motif (Figure 4A) in the NBS is likely involved in nucleotide binding in NBS-LRR proteins (Tameling et al., 2002 ) and is necessary for their function in disease resistance (Dinesh-Kumar et al., 2000; Bendahmane et al., 2002). To test the P-loop role in N self-association, we created a 35S promoter N construct with the mutation GK221,222AA. The mutant protein was stable and, as expected, failed to trigger a resistance response against TMV (Figure 4B) in either the HR or TMV:GFP resistance assay method. This mutant also lost the ability to oligomerize or coprecipitate with the wild-type protein in the presence of the elicitor (Figure 4C) and did not increase in abundance after P50 elicitation. From these results, we conclude that elicitor-mediated oligomerization and stabilization of N requires the presence of an intact P-loop motif. Furthermore, our data show that the CP does not reduce the abundance of the N protein (Figure 4C).
By contrast, a second conserved motif, RNBS-A, does not affect the coprecipitation of N, although it is required for N protein function in resistance assays. RNBS-A is located between the P-loop and kinase-2 motifs, and its consensus sequence differs between TIR and non-TIR R proteins (Meyers et al., 1999
We created a 35S-driven N construct with the RNBS-A mutation LL270,271AA (NAA; Figure 5). The same mutation in the equivalent LXXLL motif of the mammalian NOD protein CTIIA caused loss of function and affected its oligomerization ability (Sisk et al., 2001
TIR Domain Interactions
We tested the significance of the TIR interactions by mutation of TIR domain amino residues at predicted solvent-exposed sites that may play a role in signaling and protein–protein interactions (Figure 6A). All nine mutants produced stable proteins, and three of them (R24A, S80A, and P110Y) were compromised in the HR assay in tobacco leaves and in the resistance assay against TMV:GFP in N. benthamiana (Figure 6C; data not shown). In the HR assay, the R24A mutant was completely inactive and the S80A and P110Y mutants induced a very weak HR. All three mutants completely failed to induce resistance in the TMV:GFP assay. The corresponding mutants of full-length N retained their ability to oligomerize in response to the P50 elicitor (Figure 6D), although the interaction was weakest for mutant R24A. Similarly, the isolated TIR domain mutants exhibited homotypic interactions, although, as with the full-length proteins, the TIR domain of R24A was the weakest interactor (Figure 6E). These results are as predicted if the coprecipitation of full-length N is mediated by homotypic interactions of the TIR domain. They also are the predicted outcomes if oligomerization of N is an early event in the elicitor-mediated activation of the disease resistance pathway.
Here, we describe elicitor-mediated oligomerization and stabilization of the N protein as novel processes associated with resistance against TMV (Figures 2 to 4  ). The oligomerization and stabilization appear to be separate processes because they are differentially affected by mutations in the RNBS-A motif of N (Figure 5). We infer that oligomerization is functionally significant because, like the N resistance response, it is dependent on an intact P-loop motif (Figure 4). Moreover, there was a correlation between N oligomerization and the elicitation of resistance in that activation of resistance with either mutant or wild-type N was always associated with oligomerization. However, oligomerization is not sufficient to trigger the resistance response (Figure 5), and we deduce that additional interactions of oligomerized N are required for N function. In addition, because silencing of a presumed downstream signaling component in the N signaling pathway (EDS1) has no effect on either the oligomerization or the stabilization of N (Figure 4), it is likely that these are early events associated with the elicitor activation of N. A model of N oligomerization-induced activation of disease resistance is described in more detail below. The results from the mutational analysis of the TIR domain are in agreement with the proposed signaling function of the TIR. The R24A, S80A, and P110Y mutations have evidently resulted in loss of function in the signaling domain but have caused only partial loss (R24A) or no loss of the oligomerization function. This proposed separation of oligomerization and signaling functions is reinforced by our analysis of an RNBS-A mutant (Figure 5). It is also consistent with the conclusion from EDS1 and NRG1 silencing (Figure 3) that N oligomerization is an early event in the sequence of events leading to P50-elicited disease resistance.
Our finding that the silencing of SGT1 resulted in low levels of N soluble protein is consistent with, although does not prove, the possibility that SGT1 is involved in the elicitor-induced stabilization of N. SGT1 interacts with the RAR1 and HSP90 cofactors of disease resistance, and it has been proposed that RAR1 and SGT1 are cochaperones of HSP90 in the folding of R proteins (Shirasu and Schulze-Lefert, 2000
However, R protein stabilization, SGT1 function, and disease resistance are not always associated in the same way. The RPM1 NBS-LRR protein, for example, which confers resistance against Pseudomonas syringae strains, is unlike N in that it is destabilized when resistance is elicited (Boyes et al., 1998 A model of N activation is shown in Figure 7. The initial events are changes to the conformation of N. We considered the possibility that these changes could be analogous to the disruption of CC and the LRR domain interactions that are associated with the elicitation of Rx-mediated resistance (Figure 7, left branch). However, in an extensive analysis (see Supplemental Tables 2 and 3 online), there was no evidence for intramolecular interactions by coexpression of N domains. Perhaps the intramolecular interactions in N are weaker than in Rx and are not effective for proteins expressed in trans. Alternatively, it is possible that elicitor-mediated activation of N involves interactions with other as yet unidentified proteins or, perhaps, a change in subcellular localization of the protein (Figure 7, right branch).
Our model proposes that these initial conformational changes would cause N to oligomerize in complexes that are required for the activation of the EDS1-dependent and perhaps other response pathways, leading to virus resistance and HR (Figure 7). In principle, the TIR interactions (Figure 6) could mediate the oligomerization process if they are exposed by the elicitor-induced changes to N. Presumably, the isolated TIR domains, being free of the rest of the N protein, would not be masked by a subcellular location or other domains in N and would be available to interact even in the absence of elicitor. We show the homotypic N interactions in Figure 7 as being direct, but we emphasize that they could be indirect and dependent on host factors that have not yet been identified. It is also possible that domains of N, in addition to the TIR, may be involved in the oligomerization process. It is possible that, as with Toll and TLRs, the TIR domain interactions are secondary to oligomerization at other more C-terminal domains (Xu et al., 2000 ; Hu et al., 2004; Sun et al., 2004). Unfortunately, the isolated NBS domains of N were not stable in the transient assay, and we could not assay their potential for homomeric interactions.
How could oligomerization of N activate the virus resistance and HR pathways? It is unlikely to be simple induced proximity of TIR domains, because the isolated TIR domains interact but do not induce an HR. Accordingly, as with other members of the NOD family, expression of the N-terminal domain alone does not activate response pathways (Inohara and Nunez, 2003
The elicitor-induced oligomerization and stabilization of N, being upstream of EDS1 (Figure 3), are the earliest identified molecular features of the N resistance pathway; therefore, they are useful for positioning other processes in the sequence of recognition and response mechanisms. Here, for example, we have shown that the CC-NBS-LRR protein NRG1 is likely to act downstream of the elicitor recognition process because elicitor-induced oligomerization/stabilization of N occurs in the NRG1-silenced plants. As overexpression of NRG1 induces responses that are not dependent on EDS1 (Peart et al., 2005
We were able to detect the N oligomerization in our transient assay system because the elicitor-induced cell death is absent. In other experimental systems, including the Rx CC-NBS-LRR protein, the elicitor-induced cell death is rapid and prevented us from detecting the transiently expressed proteins (Moffett et al., 2002
Plant Material Wild-type Nicotiana benthamiana, N-transgenic N. benthamiana line 310A (Bendahmane et al., 1999 ), tobacco (Nicotiana tabacum) cv Samsun (NN), and tobacco cv Petite Havana (nn) plants were grown in glasshouses under controlled light and temperature.
Plasmid Construction To clone N fragments into pHAN and pMYCN, we amplified the different fragments from an N genomic clone (see below) using forward and reverse primers carrying XhoI and AvrII restriction sites, respectively. The amplified products were cloned directionally into pHAN and pMYCN digested with XhoI and AvrII. A list of the different clones obtained, the N sequences they comprise, and the primers used for amplification is shown in Supplemental Table 1 online. Primer sequences are available on request. All PCRs were performed using Pfu polymerase, and the identities of all clones obtained were confirmed by sequencing. Full-length HA-tagged N (35S:N:HA) consists of the complete N genomic sequence (from start to stop codons) plus the HA tag under the control of the CaMV 35S promoter and terminator. It was built from several N fragments as follows. pHAN:LRR1 and pHAN:LRR2 were digested with BamHI. The insert derived from pHAN:LRR1 and the backbone derived from pHAN:LRR2 were gel-purified and ligated to obtain pHAN:LRR4. Next, this construct and pHAN-TNBL were digested with SalI and DraIII. The insert derived from pHAN:TNBL and the backbone derived from pHAN:LRR4 were gel-purified and ligated to obtain 35S:N:HA. Exactly the same procedure, but with pMYCN-based constructs, was used to create 35S:N:MYC.
To place MYC-tagged N under the control of the N native promoter (gNMYC), a 12.3-kb DNA fragment containing N coding sequence, introns, 4.3 kb of 5' flanking sequence, and 1.3 kb of 3' flanking sequence was transferred from plasmid pTG34 (Whitham et al., 1994 All mutants of N described in this work were obtained by PCR methods. Forward N primers starting at ATG and including an XhoI site (primer Nup) were used with reverse primers including the mutation, whereas forward primers including the mutation were used together with a downstream reverse primer (NBSdw; see Supplemental Table 1 online). Both PCR fragments were gel-purified, subjected to five cycles of PCR without primers, and then amplified with primers Nup and NBSdw. The final amplified fragment contained a StuI site that was always located downstream of the mutation. The PCR product was digested with XhoI and StuI and replaced into 35S:N:HA and 35S:N:MYC. The sequences of primers used for mutagenesis are available on request. All PCRs were performed using Pfu polymerase, and the identities of all clones obtained were confirmed by sequencing. Mutants in the context of the TIR domain were obtained by PCR methods. Forward N primers starting at ATG and including an XhoI site (primer Nup) were used with reverse primers including the mutation, whereas forward primers including the mutation were used together with a downstream reverse primer (TIRdw; see Supplemental Table 1 online). Both PCR fragments were gel-purified, subjected to five cycles of PCR without primers, and then amplified with primers Nup and TIRdw. The PCR product was digested with XhoI and AvrII and cloned directionally into pHAN and pMYCN.
To obtain 35S:NRG1:HA, the NRG1 sequence was amplified from a cDNA clone (Peart et al., 2005
The sequence of the P50 elicitor (nucleotides 2082 to 3418 from TMV, which correspond to the helicase domain of the viral replicase) (Erickson et al., 1999
To obtain P50:GFP, the same sequence described above was amplified with forward and reverse primers with SalI and XbaI restriction sites, respectively. The resulting product was digested and cloned into a pBINY53-derived binary vector containing the GFP4 sequence (P. Mestre, unpublished data). Primer sequences are available upon request. The other constructs used in this work have been described elsewhere: CP (Mestre et al., 2000
Agrobacterium tumefaciens–Mediated Transient Expression
Protein Extraction, Immunoprecipitation, and Immunoblotting
Virus-Induced Gene Silencing
Sequence Analysis
Accession Numbers
Supplemental Data
We thank Peter Moffett for advice on immunoprecipitation techniques, Jack Peart for advice on experiments using TMV:GFP, Thomas Lahaye for providing Bs4TIR:MYC, and Michal Swiderski for providing Rps4TIR:HA. We are grateful to Scott Peck and Ken Shirasu for critical reading of the manuscript. Recombinant viruses were contained in greenhouses under the Department for Environment, Food, and Rural Affairs license PHL 161A/4391. This work was supported by the Gatsby Charitable Foundation and the Biotechnology and Biological Sciences Research Council (Grant 83/P15172).
1 Current address: Unité Mixte de Recherche, Santé de la Vigne et Qualité du Vin, Institut National de la Recherche Agronomique/Université Louis Pasteur, Laboratoire de Génétique et d'Amélioration de la Vigne BP507, 28 Rue de Herrlisheim, F-68021 Colmar Cedex, France.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: David C. Baulcombe (david.baulcombe{at}sainsbury-laboratory.ac.uk).
[W] Online version contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.037234. Received August 19, 2005; Revision received November 15, 2005. accepted December 1, 2005.
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-HA) or anti-myc (IP: 
