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. 2009 Jun 5;284(23):15867-79.
doi: 10.1074/jbc.M900519200. Epub 2009 Apr 3.

A family of bacterial cysteine protease type III effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes

Robert H Dowen  1 , James L Engel, Feng Shao, Joseph R Ecker, Jack E Dixon
Affiliations

A family of bacterial cysteine protease type III effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes

Robert H Dowen et al. J Biol Chem. .

Abstract

Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like effectors, ORF4 and NopT, autoproteolytically process following delivery into the plant cell to expose embedded sites for fatty acylation. Host-dependent lipidation of these three effectors directs plasma membrane localization and is required for the avirulence activity of AvrPphB. Surprisingly, the AvrPphB-like effectors RipT, HopC1, and HopN1 utilize an acylation-independent mechanism to localize to the cellular plasma membrane. Although some AvrPphB-like effectors employ acylation-independent localization strategies, others hijack the eukaryotic lipidation machinery to ensure plasma membrane localization, illustrating the diverse tactics employed by type III effectors to target specific subcellular compartments.

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Figures

FIGURE 1.
FIGURE 1.
Multiple amino acid sequence alignment of the AvrPphB family reveals conserved residues within the NH2 terminus. A, members of the AvrPphB family were identified by PSI-BLAST using the AvrPphB sequence as the query and the amino termini were aligned. The full alignment is displayed in supplemental Fig. S1. Residues that share homology, as well as the catalytic cysteine and the embedded acylation sites, are colored according to the key. The known autoprocessing site in AvrPphB is also indicated (Shao et al. (12) and Puri et al. (15)). Proteins examined in this study are in bold. Additional accession numbers are as follows: AvrPphB, Q52430; HopAW1, AAX12112; ORF4, AAD47206; AvrPpic2, CAC16701; HopC1, AAO54131; HopN1, AAO54892; RipT, NP_521333; NopT, AAB91961; Blr2058, NP_768698; Blr2140, NP_768780. B, a schematic of the full-length effector proteins examined in this study. The catalytic residues, autoprocessing sites, and acylation sites are displayed according to the key.
FIGURE 2.
FIGURE 2.
Additional AvrPphB family members undergo autoproteolytic cleavage at specific residues. A, the indicated proteins were in vitro transcribed and translated in the presence of [35S]methionine and aliquots were removed at the indicated time points. The samples were subjected to SDS-PAGE and proteins were visualized by autoradiography (Unprocessed proteases, U; mature proteases, M). Secondary start methionines produce additional protein species during translation (asterisks) that generate the following proteins: AvrPphB, M = 22 kDa and * (from Met-57) = 23 kDa; ORF4, M = 23 kDa and * (from Met-51) = 30 kDa; HopC1, U = 29 kDa and * (from Met-29) = 26 kDa. The experiment was performed three times with similar results. B, the autoprocessing sites of the mature recombinant proteins were determined by Edman degradation (footnote a data from Puri et al. (15); footnote b data from this study; footnote c data from Dai et al. (47)). The three residues that precede the cleavage site are shown in green. C, sequence alignment of the known autoproteolytic processing site in AvrPphB and the cleavage site in PBS1 with the three conserved amino acids that precede the cleavage sites shown in green. Mutation of these residues to alanine (red) in PBS1 inhibits cleavage by AvrPphB (Shao et al. (11)). D, the indicated P1/P2/P3 triple mutants were generated and analyzed as described in A. Residues that allow autoproteolytic processing are shown in green and mutant residues that prevent cleavage are colored in red. The catalytically inactive mutants (C/S) are deficient in autoprocessing activity. Additional protein species generated from secondary start methionines (AvrPphB and ORF4) are as described in A. Each experiment was repeated twice with similar results.
FIGURE 3.
FIGURE 3.
Autoproteolytic processing of AvrPphB family members results in N-myristoylation and S-palmitoylation of the new amino terminus. A, the NH2-terminal sequences of the autoprocessed, mature proteins were examined for eukaryotic acylation consensus sites. Important residues are colored according to the key. The myristoylation consensus sequence is based on previous experiments (Utsumi et al. (28)). Full-length wild-type and mutant effectors were expressed in S. cerevisiae in the presence of 30 μCi/ml [3H]myristic acid (B) or 50 μCi/ml [3H]palmitic acid (C). After labeling for 4 h, the FLAG-tagged effectors were immunoprecipitated, subjected to SDS-PAGE, and analyzed by autoradiography (top panel) or Western blotting with an anti-FLAG antibody (bottom panel). An asterisk indicates nonspecific bands. Yeast radiolabeling experiments were performed twice with similar results.
FIGURE 4.
FIGURE 4.
Acylated and non-acylated AvrPphB family members are differentially associated with S. cerevisiae membranes. A, strains carrying the indicated FLAG-tagged effectors or empty vector (V) were induced with galactose for 8 h and homogenized. Total extracts (T) were fractionated into soluble (S) fractions and insoluble membrane pellets (P) by ultracentrifugation at 100,000 × g. Equal volumes of each fraction were subjected to SDS-PAGE, blotted, and probed with anti-FLAG or anti-v-H-Ras (plasma membrane marker) antibodies. B, membranes were isolated as in A and resuspended in control lysis buffer, high salt buffer (1 m NaCl, 10 mm Tris-HCl, pH 7.4), denaturing buffer (2 m urea, 10 mm Tris-HCl, pH 7.4), high pH buffer (0.1 m Na2CO3, pH 11.5), or buffer containing detergent (1% Triton X-100, 10 mm Tris-HCl, pH 7.4). Treated samples were re-ultracentrifuged and equal volumes of the soluble (S) and pellet (P) fractions were subjected to SDS-PAGE and Western blot (WB) analysis as in A. Each experiment was performed twice with similar results.
FIGURE 5.
FIGURE 5.
AvrPphB-like family members localize to the plasma membranes of Chinese cabbage cells. COOH-terminal-tagged YFP effector proteins were transiently expressed in Chinese cabbage epidermal cells using particle bombardment. Representative fluorescent images of cells expressing wild-type effectors or acylation-deficient mutants are indicated. Control bombardments were performed using the cytosolic YFP or plasma membrane-localized PIP2A (plasma membrane intrinsic protein 2A)-CFP proteins. Both YFP and CFP fluorescence are colored in green. Bar, 50 μm.
FIGURE 6.
FIGURE 6.
Host acylation of AvrPphB is required for avirulence activity in Arabidopsis plants carrying PBS1. A, adult Col-0 leaves were syringe infiltrated (opposite to the marked leaf half) with ∼3.75 × 107 cfu/ml P. syringae pv. tomato DC3000 (Pst) or P. fluorescens pLN1965 (Pf) strains expressing AvrPphB or ORF4. Also, plants were inoculated with 10 mm MgCl2 (Mock) or strains carrying the pVSP61 empty vector (EV). Ratios below each leaf indicate the number of HR positive leaves/total number of leaves inoculated. B, transgenic pbs1–1:PBS1-HA plants were inoculated as in A with the indicated Pst strains. Leaf tissue was harvested 14 h.p.i., homogenized, and 10 μg of total protein was subjected to SDS-PAGE. Blots were analyzed by anti-HA Western blotting (WB). Three individual T2 plants were assayed for each infection condition and produced identical results. C, Col-0 or pbs1-1 plants were inoculated as described in A with Pst strains carrying the indicated avrPphB alleles (Gly-63, myristoylation site; Cys-64, palmitoylation site; Cys-98, catalytic cysteine). Data were collected 20 h.p.i. and are representative of two independent experiments. D, Pst strains carrying the indicated alleles were grown in Hrp-inducing minimal media. Cultures were partitioned into cell-bound and secreted fractions by centrifugation. Protein samples were subjected to SDS-PAGE, blotted, and probed with antibodies against AvrPphB or NPTII (control for nonspecific lysis). E, Arabidopsis seedlings were inoculated by dipping with Pst strains (∼2.5 × 107 cfu/ml) carrying the indicated effector alleles. At day 0 (white bars) or day 3 (black bars) the bacteria were extracted and quantified. Data are represented as the mean ± S.E. of four technical replicates. The experiment was repeated twice with similar results. F, Arabidopsis seedlings were inoculated as described in E with the indicated Pst strains. Tissue was harvested 24 h.p.i. and RNA was subjected to reverse transcriptase-qPCR analysis using PR1 and TUBULIN3 specific primers. PR1 mRNA levels (relative to TUB3) were calibrated to mock-treated samples (2−ΔΔCt). Data are represented as the mean ± S.E. of at least 5 biological replicates from two independent experiments.
FIGURE 7.
FIGURE 7.
A model for the subcellular localization strategies of the AvrPphB-like effector proteins in the plant cell. The indicated strains are shown in gray, the TTSS in purple, and the effectors in black. Effectors are classified according to their ability to self-proteolytically process (red star in model), to be acylated by the host (N-myristoylation, orange; S-palmitoylation, green), and their biological function. AvrPphB, ORF4, and NopT are lipidated by the host machinery (NMT, N-myristoyl transferase; PAT, palmitoyl acyltransferase), whereas RipT, HopC1, and HopN1 are directed to the PM by an unknown mechanism. Host acylation of AvrPphB is essential for cleavage of PBS1 (blue) and initiation of RPS5 (red) defenses. Unknown targets of ORF4, NopT, and RipT are also included (blue) and contain putative target sequences.
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