Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan;164(1):340-51.
doi: 10.1104/pp.113.227686. Epub 2013 Nov 13.

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

Affiliations
Free PMC article

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

Dong Qi et al. Plant Physiol. .
Free PMC article

Abstract

The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification.

Figures

Figure 1.
Figure 1.
S-Acylation mediates PBS1 localization to the PM. A, The predicted N-terminal S-acylation motif in PBS1 is necessary and sufficient for localizing PBS1 to the PM. The indicated PBS1 derivatives were fused to sYFP and transiently expressed in N. benthamiana leaves using a dexamethasone-inducible promoter. N20 indicates the first 20 amino acids of PBS1, while n20 is the same fragment with the G2AC3/6A triple substitution, which mutates the predicted S-acylation site. Confocal laser scanning microscopy was performed at 5 h post dexamethasone induction. All images are three-dimensional projections from a Z-stack. B, PBS1 is S-acylated. Cell extracts of N. benthamiana tissue expressing N20:sYFP-HA were subjected to immunoprecipitation using α-HA matrix. Immunoprecipitates (IP) were treated with 50 mm N-ethylmaleimide to block free sulfhydryl groups, incubated with 1 m hydroxylamine to hydrolyze any Cys-palmitate thioester bonds, and then treated with 1 μm Biotin-BMCC to label free sulfhydryl groups resulting from cleaved thioester bonds. Modified immunoprecipitates were analyzed by immunoblot (IB) with streptavidin-horseradish peroxidase. C, Full-length PBS1 is palmitoylated when expressed in yeast. The indicated PBS1 constructs were expressed in yeast in the presence of [3H]palmitic acid, immunoprecipitated, and separated on an SDS-PAGE gel, and radiolabeling was detected by autoradiography. EV, Empty vector; WT, wild type. D, Engineered PBS1 cleavage products interact with each other at the PM. BiFC was detected between the N-terminal PBS1 cleavage product (nPBS1:NYFP) and the C-terminal PBS1 cleavage product (cPBS1:CYFP) but not between nPBS1:NYFP and 3× HA:CYFP used as a negative control. The indicated constructs were coexpressed in N. benthamiana leaves, and imaging was performed at 5 h post dexamethasone induction.
Figure 2.
Figure 2.
S-Acylation of PBS1 is required for RPS5-mediated resistance in Arabidopsis. A, S-Acylation of PBS1 is necessary for HR induction by P. syringae strain DC3000(avrPphB). The pbs1-1 mutant was transformed with the indicated PBS1 constructs under the control of the native PBS1 promoter and then assessed for RPS5-mediated HR by inoculation with DC3000(avrPphB). HR was scored at 16 h post inoculation. Plants transformed with empty vector were included as a negative control. B, The PBS1 S-acylation mutant is susceptible to P. syringae strain DC3000(avrPphB). The same transgenic lines were assessed for the growth of DC3000(avrPphB) following syringe infiltration. Data shown represent means and sd (n = 3). Two independent experiments were performed with similar results, and two independent transgenic lines were tested for each mutant.
Figure 3.
Figure 3.
S-Acylation of PBS1 is required for cleavage by AvrPphB in vivo. A, S-acylation of PBS1 is required for cleavage by AvrPphB delivered by DC3000(avrPphB) into plant cells. Transgenic plants expressing wild-type PBS1-HA and the G2AC3/6A mutant were syringe infiltrated with DC3000(avrPphB). The infiltrated tissues were harvested 6 h post inoculation and subjected to immunoblot analysis with anti-HA antibodies. Transgenic plants expressing wild-type PBS1 and challenged with DC3000 lacking avrPphB were employed as the negative control. B, S-Acylation of PBS1 is not structurally required for cleavage by AvrPphB. The G2AC3/6A-PBS1 mutant was transiently coexpressed with a G2A-AvrPphB mutant in N. benthamiana, which localizes to the cytoplasm. The C98S mutant of AvrPphB, which lacks protease activity, was included as the negative control. Tissues were harvested at 5 h post dexamethasone induction. Asterisks indicate C-terminal PBS1 cleavage products.
Figure 4.
Figure 4.
The EAR5 pbs1 mutants are unable to activate RPS5 in response to P. syringae strain DC3000(avrPphB). A, Schematic diagram of missense mutations identified in PBS1. The kinase domain and activation segment of PBS1 are represented by pink and dark blue boxes, respectively. Missense substitutions found in mutants are indicated above the bar and marked by asterisks. B, The EAR5 pbs1 mutants fail to induce HR following inoculation with DC3000(avrPphB). Leaves were syringe inoculated with DC3000(avrPphB) and photographed 24 h later. C, The EAR5 pbs1 mutants are susceptible to DC3000(avrPphB). Data represent means and sd. Different letters above the bars denote significant differences determined by a two-tailed Student’s t test (P < 0.01). This experiment was repeated twice with similar results.
Figure 5.
Figure 5.
The pbs1-3 and pbs1-4 mutations block cleavage by AvrPphB. A, The indicated pbs1 alleles fused to an HA tag were transiently expressed in N. benthamiana in the presence of empty vector (e.v.) or AvrPphB. All constructs were under the control of a dexamethasone-inducible promoter, and samples were prepared 6 h post dexamethasone induction. Cleavage of PBS1 was confirmed by immunoblot analysis with anti-HA antibody. Only G103R (pbs1-3) and G286D (pbs1-4) were not cleaved by AvrPphB. B, Longer exposure of the immunoblot shown in A to detect G103R. This experiment was repeated twice with similar results.
Figure 6.
Figure 6.
The C-terminal SEMPH motif discriminates between PBS1 and PBL27. A, An HR is induced by coexpression of a PBL27 engineered N-terminal cleavage product together with an engineered C-terminal cleavage product of PBS1 in the presence of RPS5. Red circles mark infiltration sites in N. benthamiana leaves. The photograph was taken 16 h after dexamethasone induction. B, Substitution of SEMPH for NARAP in PBL27 enables PBL27 to be recognized by RPS5. The indicated proteins were coexpressed in N. benthamiana together with wild-type AvrPphB (+) or its inactive C98S mutant (−) in the presence of RPS5. C, Ribbon diagram of the predicted structure of the PBS1 kinase domain based on alignment with the crystal structure of the tomato Pto kinase (Protein Data Bank accession no. 2QKW). The AvrPphB cleavage site (GDK) is marked in red. The RPS5 recognition motif (SEMPH) is denoted in light green. New PBS1 mutations pbs1-3 (G103R), pbs1-4 (G286D), pbs1-5 (G98E), and pbs1-6 (S244F) are depicted as amino acid substitutions according to their positions within the molecule (orange). The P-loop is shown in turquoise and Lys-115 is shown in blue. Supplemental Movie S1 shows this diagram rotating in space.

Similar articles

See all similar articles

Cited by 19 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback