Abstract
Nucleotide-binding leucine-rich repeat receptors (NLRs) are the most abundant type of immune receptors in plants and can trigger a rapid cell-death (hypersensitive) response upon sensing pathogens. We previously cloned the wheat NLR Sr35, which encodes a coiled-coil (CC) NLR that confers resistance to the virulent wheat stem rust race Ug99. Here, we investigated Sr35 signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. Expression of Sr35 in N. benthamiana leaves triggered a mild cell-death response, which is enhanced in the autoactive mutant Sr35 D503V. The N-terminal tagging of Sr35 with green fluorescent protein (GFP) blocked the induction of cell death, whereas a C-terminal GFP tag did not. No domain truncations of Sr35 generated cell-death responses as strong as the wild type, but a truncation including the NB-ARC (nucleotide binding adaptor) shared by APAF-1, R proteins, and CED-4 domains in combination with the D503V autoactive mutation triggered cell death. In addition, coexpression of Sr35 with the matching pathogen effector protein AvrSr35 resulted in robust cell death and electrolyte leakage levels that were similar to autoactive Sr35 and significantly higher than Sr35 alone. Coexpression of Sr35-CC-NB-ARC and AvrSr35 did not induce cell death, confirming the importance of the leucine-rich repeat (LRR) domain for AvrSr35 recognition. These findings were confirmed through Agrobacterium-mediated transient expression in barley. Taken together, these results implicate the CC-NB-ARC domains of Sr35 in inducing cell death and the LRR domain in AvrSr35 recognition.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.