Affiliation:
1. Crop Production and Pest Control Research Unit, U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), West Lafayette, IN 47907
2. Department of Biological Sciences, University of North Carolina, Charlotte, NC 28223
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 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 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 coexpressed with AvrPphB, indicating that the individual domains and fragments of PBR1 are insufficient to trigger HR. Intriguingly, introducing the typically autoactivating D496V mutation within NB-ARC-containing fragments of PBR1 does not activate immune signaling, revealing that PBR1-mediated immune signaling requires the cooperation of all domains in cis. Using coimmunoprecipitation 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 extend our understanding of NLR-mediated immune signaling. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Funder
Agricultural Research Service
U.S. Wheat and Barley Scab Initiative