Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein

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.

Figure 1.

The RPS2 CC domain can partially substitute for the RPS5 CC domain. The indicated constructs were transiently coexpressed in N. benthamiana, and leaves were detached for photography 24 h post DEX induction. D266E indicates full-length RPS5 with the D266E substitution. 2-5-5 indicates the chimeric construct with the RPS2 CC domain and the RPS5 NBS-LRR domains. C98S indicates the protease-inactive mutant form of AvrPphB. A, The 2-5-5 chimera can induce HR in response to PBS1 cleavage. B, The 2-5^(D266E)-5 chimera is not autoactive but can induce HR in response to PBS1 cleavage. C, Deletion of the LRR domain from the 2-5^(D266E)-5 chimera, but not the 2-5-5 chimera, causes autoactivation.

Figure 2.

The RPS5 N-terminal acylation motif is required for RPS5-mediated HR. The indicated constructs were transiently coexpressed in N. benthamiana leaves as described in Figure 1. A, Modification of the N-terminal myristoylation motif (G2/3A) disables D266E autoactivation but not the recognition of PBS1 cleavage. B, Modification of the N-terminal palmitoylation residue (C4A) produces the same phenotype as the G2/3A substitution. C, Mutations of both myristoylation and palmitoylation residues (G2/3AC4A) completely disable RPS5-mediated HR in the presence of PBS1 and AvrPphB. D, Quantification of RPS5-mediated cell death in N. benthamiana by measurement of electrolyte leakage. sYFP was included as the negative control.

Figure 3.

The N-terminal acylation motifs of the RPS5 CC domain are not required for Co-IP with PBS1 or with the NBS domain. G2/3AC4A substitutions in the predicted RPS5 N-terminal acylation residues do not abolish Co-IP between the RPS5 CC domain and PBS1 (left panels) or the RPS5 CC domain and the NBS domain (right panels). All constructs were transiently expressed in N. benthamiana. Empty vector (EV) was employed as the negative control. IB, Immunoblot; IP, immunoprecipitation.

Figure 4.

The N-terminal acylation motifs are required for the localization of RPS5 to the PM and for protein stabilization. A, RPS5 is localized to the PM. Mutating the RPS5 myristoylation and palmitoylation motifs individually slows the accumulation of RPS5-sYFP at the PM, while modification of both together eliminates PM accumulation. The indicated constructs were transiently coexpressed in N. benthamiana leaves and imaged using confocal laser scanning microscopy at the indicated times following DEX induction. Free mCherry was included as a reference for cytoplasmic localization (C) and nuclear localization (N). B, Modification of the myristoylation and palmitoylation motifs of RPS5 causes reduced protein accumulation. Coomassie Brilliant Blue staining was included as a control to show equal loading. Samples were prepared at 6 or 24 h post DEX induction and loaded as indicated.

Figure 5.

The N-terminal 20 amino acids of RPS5 are sufficient for directing RPS5 to the PM. The mCherry column shows free mCherry, while the sYFP column shows the indicated fusion proteins. Constructs were transiently coexpressed in N. benthamiana leaves and imaged 8 h following DEX induction. N indicates the nucleus.

Figure 6.

The RPS5 LRR domain is required for the recognition of PBS1 cleavage. A, Deletion of the LRR domain in the RPS5 G2/3A or G2/3A-D266E background does not cause autoactivation but eliminates the recognition of PBS1 cleavage. B, Swapping the RPS2 LRR domain for the RPS5 LRR domain causes autoactivation. The indicated constructs were transiently coexpressed in N. benthamiana leaves, and leaves were photographed 24 h post DEX induction.

Figure 7.

Deletion analysis of the RPS5 LRR domain. A, Diagram of RPS5 LRR C-terminal deletion mutants. RPS5 CC, NBS, and LRR domains are indicated by dashed boxes, solid boxes, and striped boxes, respectively. CC-NBS-13LRR indicates wild-type RPS5 with the intact LRR domain containing 13 LRR repeats. B, Electrolyte leakage analysis of N. benthamiana leaf discs transiently expressing the indicated constructs. C and D, Photographs of N. benthamiana leaves taken 24 h post DEX induction of the indicated constructs.

Figure 8.

Internal LRR deletions block the recognition of PBS1 cleavage but not autoactivation by the D266E substitution. The indicated constructs were transiently expressed in N. benthamiana. Photographs were taken 24 h post DEX induction.

Figure 9.

Model for the regulation of RPS5 activation. PBS1 associates with the CC domain at the PM in a preactivation complex in which the LRR domain traps the ADP nucleotide, keeping RPS5 in the off state. The AvrPphB effector cleaves PBS1, causing a conformational change in PBS1 that then binds to the LRR domain of RPS5, causing the LRR:NBS interface to open. This promotes nucleotide exchange, leading to a conformational change in RPS5, enabling RPS5 to activate the HR.