Analysis of cell death induction by the barley NLR immune receptor PBR1

Authors: Jaiswal, Namrata; MYERS, ARIANA - US Department Of Agriculture (USDA); Cameron, Terri; CARTER, MORGAN - University Of North Carolina; Scofield, Steven - Steve; Helm, Matthew

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2023
Publication Date: 11/27/2023
Citation: Jaiswal, N., Myers, A., Cameron, T.L., Carter, M.E., Scofield, S.R., Helm, M.D. 2023. Analysis of cell death induction by the barley NLR immune receptor PBR1. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-01-23-0005-R.
DOI: https://doi.org/10.1094/PHYTOFR-01-23-0005-R

Interpretive Summary: Plants have a sophisticated immune system capable of detecting and conferring resistance to plant pathogens. This system is composed of resistance proteins whose function is to detect molecules that the pathogen secretes, known as effectors, into the plant cell to aid infection. If the resistance protein is capable of recognizing the effector , it will trigger a robust resistance response. Very little is known about how resistance proteins perceive effectors and activate immune responses. Here, we tested the functions of the 3 major domains of the PBR1 barley resistance protein and found that, unlike some other resistance proteins, they all must be present to activate resistance. These results significantly advance our understanding of how a barley disease resistance protein detects pathogens and opens the door for engineering new disease resistances in crop plants.

Technical Abstract: 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. We found that co-expression of PBR1 with AvrPphB resulted in robust cell death in N. benthamiana, confirming previous observations that PBR1 is indeed the cognate NLR that recognizes AvrPphB. The N-terminal tagging of PBR1 with super Yellow Fluorescent Protein (sYFP) abolished PBR1-mediated cell death. Furthermore, none of the individual protein domains or truncations 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 demonstrating PBR1-mediated immune signaling requires cooperation of all domains. 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, the findings presented herein provide valuable insights into PBR1-mediated disease resistance and extends upon our understanding of NLR-mediated immune signaling.