The Interaction Proteome of the Plant NLR Immune Receptor 'N'

Plants rely on a complex and multi-layered immune system to detect and launch defenses against pathogens spanning many kingdoms. Plant cells are patrolled by a diverse group of immune receptors bearing a nucleotide-binding domain (NB) and a leucine-rich repeat domain (LRR), which are hence called NLRs. NLRs trigger an impressive array of defense signaling events including induction of an oxidative burst, ion fluxes, production of salicylic acid, activation of MAPK cascades, transcriptional reprogramming, and oftentimes localized programmed cell death at the site of pathogen invasion. One of the best studied NLRs is the N immune receptor in Nicotiana, which confers resistance to Tobacco mosaic virus. N is functional in the model plant Nicotiana benthamiana, which was recently sequenced, is amenable to transient protein expression by agroinfiltration, and permits gene functional analysis by virus-induced gene silencing (VIGS). Therefore, N is an appealing model for the study of NLRmediated defense. NLR immune receptors are thought to reside in large protein complexes, and identifying members of these complexes has been a priority in the field of plant defense. This goal, however, has been elusive because NLRs are low abundance proteins that are recalcitrant to over-expression. Recent improvements in affinity purification, mass spectrometry instrumentation, and bioinformatics have made affinity purification-mass spectrometry (AP-MS) a tractable approach for characterizing NLR-containing complexes. Here we performed AP-MS of N in its resting and activated states and identified several known and many previously unreported N-associated proteins. These proteins shed new light on how N may detect TMV, switch into its activated state, and induce several key defense signals. We unexpectedly found that N-associates with several membrane proteins that have known, positive roles in programmed cell death, and showed that N is partially membrane-associated. We also found that support for N association with HSP90 is lost upon activation, suggesting that dissociation from HSP90 accompanies N activation. A second aim of this study was to investigate N nuclear transport. We discovered that a previous report of N nuclear localization was inaccurate due to a technical artifact. We then found that N is indeed partially nuclear, but not to the extent previously envisaged. We also observed activated N in cytoplasmic subcellular bodies. These findings provide a broad illustration of N complexes before and after activation and directly implicate many new proteins and processes in N-mediated defense.