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). In addition, at least with TIR R proteins, it seemed possible, based on a comparison with Toll-like receptors (TLRs) and members of the interleukin-1 receptor superfamily (IL-1Rs), that the N-terminal domains would oligomerize. TLRs and IL-1Rs are transmembrane receptors with a cytoplasmic TIR domain and extracellular LRR and Ig domains, respectively (Silverman and Maniatis, 2001). After interaction with an extracellular ligand, these receptor proteins oligomerize; in turn, there are homotypic protein–protein interactions between the intracellular TIR domains. Activation of the signaling pathway follows from the oligomerization of the TIR domains (Xu et al., 2000; Hu et al., 2004; Sun et al., 2004).
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). Interactions of the CC and LRR domains were detected, but they were heterotypic: CC interacted with the NBS-LRR domains, whereas LRR interacted with CC-NBS (Moffett et al., 2002). Moreover, these interactions were disrupted rather than induced by the elicitor. Based on these findings, it was proposed that the activation of Rx involves conformational changes, as with NOD proteins, but that oligomerization was not required.
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). It mediates recognition of the helicase domain in the tobacco mosaic virus (TMV) replicase (Erickson et al., 1999) and activates a resistance response requiring several known general cofactors of disease resistance: ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) (Peart et al., 2002a), SUPPRESSOR OF G-2 ALLELE OF SKIP1 (SGT1) (Peart et al., 2002b), REQUIRED FOR Mla12 RESISTANCE1 (RAR1) (Liu et al., 2002b), HEAT SHOCK PROTEIN90 (HSP90) (Liu et al., 2004a; Lu et al., 2003), members of the COP9 signalosome (Liu et al., 2002a), and protein kinases (Jin et al., 2003; Liu et al., 2004b). Additionally, N REQUIREMENT GENE 1 (NRG1), a CC-NBS-LRR protein, has been shown to be specifically involved in the N-mediated response (Peart et al., 2005). Using transient expression of epitope-tagged proteins, we show that early events in the pathway leading to TMV resistance are oligomerization and stabilization of the N protein. Based on these results, we propose that elicitor-mediated activation of N, and possibly of other NBS-LRR proteins, is similar to the ligand-mediated triggering of NOD proteins.
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