Membrane Proteins Daily

Plant Physiol. 2012 Feb 13;
Qi D, Deyoung BJ, Innes RW

The Arabidopsis 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 (sYFP) fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine 2) and palmitoylation residues (cysteine 4) 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 sYFP to the PM. C-terminal truncations of RPS5 revealed that the first 4 LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in autoactivation of RPS5, indicating that the LRR domain must co-evolve 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.

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Structure-Function Analysis of the Coiled-Coil and Leucine-Rich Repeat Domains of the RPS5 Disease Resistance Protein.