The barley (Hordeum vulgare subsp. vulgare) disease resistance protein AvrPphB Response 1 (PBR1) mediates recognition of the Pseudomonas syringae effector, AvrPphB. PBR1 belongs to the coiled-coil nucleotide-binding leucine-rich repeat (CNL) family. However, little is known about the molecular mechanisms that lead to PBR1-dependent cell death (hypersensitive reaction; HR) in response to AvrPphB. Here, we investigated PBR1 immune signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. The N-terminal tagging of PBR1 with super Yellow Fluorescent Protein (sYFP) abolished PBR1-mediated cell death, demonstrating that an N-terminal epitope tag disrupts PBR1-mediated immune signaling. Furthermore, none of the individual protein domains (CC, NB-ARC, and LRR) or truncations (CC—NB-ARC and NB-ARC—LRR) of PBR1 induced a HR-like cell death response as strong as full-length PBR1 when co-expressed with AvrPphB, indicating that the individual domains and fragments of PBR1 are insufficient to trigger HR. Intriguingly, introducing the typically auto-activating D496V mutation within NB-ARC-containing fragments of PBR1 does not activate immune signaling revealing PBR1-mediated immune signaling requires cooperation of all domains in cis. Using co-immunoprecipitation and split-luciferase assays, we also show full-length PBR1 self-associates in the absence of AvrPphB and such self-association is not dependent on a functional P-loop/Walker A motif. Collectively, these findings provide valuable insights into PBR1-mediated disease resistance and extends upon our understanding of NLR-mediated immune signaling.