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.
Highlights
Transient overexpression of wheat Sr35 with a C-terminal green fluorescent protein (GFP) tag driven by the 35S promoter in Agrobacterium-infiltrated leaves of N. benthamiana resulted in a weak cell-death response that was sometimes difficult to detect macroscopically but was always confirmed in the more sensitive electrolyte leakage experiments described below
The results presented in this study provide evidence that Sr35 is capable of inducing cell death in the presence of the matching AvrSr35 effector in both N. benthamiana and barley
The difference between the mild cell-death response induced by the wild-type Sr35 construct (Fig. 1B) and the strong celldeath response observed with the coinfiltration of Sr35 and AvrSr35 (Fig. 5B) suggests that the wild-type Sr35 protein is present mainly in an inactive state and can be activated by the AvrSr35 effector in a heterologous system
Summary
Overexpression of Sr35 triggers cell death in Nicotiana benthamiana. In order to investigate Sr35 signaling, we sought to develop a system in which we could rapidly assess defense signaling from the Sr35 receptor. The wild-type constructs (with and without the mutations at the putative palmitoylation sites) showed significantly higher leakage levels than the empty vector and K206R P-loop mutant in both single experiment and combined ANOVA analyses (more than threefold higher, Tukey test, P < 0.01) (Supplementary Table S5). Sr35 CC-NB-ARC D503VGFP did not induce as strong a cell-death response in barley as in N. benthamiana, but several of the infiltrated leaves (four of 20) showed signs of macroscopic cell death (Fig. 4), and this result was reproducible between experiments This could be due to differences in protein expression levels between the two systems, because the 35S promoter used in these experiments is more effective in dicot than in monocot species (Christensen et al 1992). We show that Sr35 recognition of AvrSr35 and the subsequent induction of cell death is dependent on the presence of the LRR domain and is blocked by a GFP tag on the N terminus of the Sr35 protein
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