Abstract
The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Here, we present a structure-function analysis of the CC and LRR domains of RPS5 using transient expression assays in Nicotiana benthamiana. We found that substituting the CC domain of RPS2 for the RPS5 CC domain did not alter RPS5 specificity and only moderately reduced its ability to activate programmed cell death, suggesting that the CC domain does not play a direct role in the recognition of PBS1 cleavage. Analysis of an RPS5-super Yellow Fluorescent Protein fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine-2) and palmitoylation (cysteine-4) residues affected RPS5 PM localization, protein stability, and function in an additive manner, indicating that PM localization is essential to RPS5 function. The first 20 amino acids of RPS5 were sufficient for directing super Yellow Fluorescent Protein to the PM. C-terminal truncations of RPS5 revealed that the first four LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for the recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in the autoactivation of RPS5, indicating that the LRR domain must coevolve with the NBS domain. We conclude that the RPS5 LRR domain functions to suppress RPS5 activation in the absence of PBS1 cleavage and promotes RPS5 activation in its presence.
Highlights
The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB
To evaluate the role of the RPS5 CC domain in the recognition of PBS1 cleavage and induction of the hypersensitive response (HR), we swapped the CC domain of RPS5 with the CC domain of RPS2, which mediates the recognition of a different P. syringae effector, AvrRpt2 (Bent et al, 1994)
Roles of the CC and leucine-rich repeat (LRR) Domains of RPS5 ilarity (45%) to RPS2 among the Arabidopsis R proteins of known function. This is consistent with a previous phylogenetic analysis that assigned RPS2 and RPS5 to the same clade distinguished by the lack of an EDVID motif found in the CC domain of most other CC-nucleotide-binding site (NBS)-LRR proteins (Meyers et al, 1999; Rairdan et al, 2008)
Summary
The Arabidopsis (Arabidopsis thaliana) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Upon detection of PAMPs, these receptors activate multiple signaling pathways, including a mitogen-activated protein kinase cascade. Successful pathogens can suppress PAMP-triggered immunity by delivering effector proteins into host cells to block signaling pathways at various steps (Desveaux et al, 2006; Kamoun, 2006; Dodds et al, 2009). This has led to the evolution of a second tier in the plant immune. The molecular mechanism regulating downstream signaling by CCNBS-LRR proteins remains unclear
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