A Bacterial Inhibitor of Host Programmed Cell Death Defenses Is an E3 Ubiquitin Ligase

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Science  13 Jan 2006:
Vol. 311, Issue 5758, pp. 222-226
DOI: 10.1126/science.1120131


The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin–conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

Type III secretion systems (T3SS) translocate bacterial virulence proteins into host cells to modulate diverse eukaryotic biochemical processes (15). Pseudomonas syringae pathovar (pv.) tomato DC3000 causes disease in tomato and Arabidopsis and uses a T3SS to evade the host's programmed cell death (PCD) response, which sacrifices a limited portion of the plant to protect the rest from systemic infection (6). The Pseudomonas syringae type III effector AvrPtoB is delivered into plant cells, where it elicits a host response that varies for resistant and susceptible tomato lines (7). In resistant plants, AvrPtoB is recognized by the Pto resistance (R) protein and elicits PCD that limits pathogen spread, which results in host immunity. In susceptible plants, however, AvrPtoB suppresses PCD associated with plant immunity, which allows the pathogen to multiply in the host and to cause disease. AvrPtoB targets a conserved PCD pathway, because AvrPtoB inhibits PCD induced by diverse agonists in plants (such as mouse BAX) and also suppresses PCD in yeast (8). The primary amino acid sequence of AvrPtoB has provided no clues about its possible function.

An acidic, C-terminal domain (CTD) of AvrPtoB, spanning residues 436 to 553, forms a protease-resistant, soluble, and stable recombinant construct that is amenable to structural determination (Fig. 1A) [figs. S1 to S3; (9)]. The structure of this domain reveals a globular fold centered on a four-stranded β sheet that packs against two helices on one face and has three very extended loops connecting the elements of secondary structure (Fig. 1B). A large and partially disordered N-terminal region spanning residues 436 to 475 packs loosely against a more tightly packed “core fold” that consists of a three-stranded β sheet packed against an α helix. The molecular surface of the AvrPtoB CTD is notable for several localized regions of differing electrostatic potential. One face of the molecule in particular shows a large basic patch sandwiched between two large acid patches (Fig. 1C).

Fig. 1.

Overall structure of the AvrPtoB CTD domain. (A) Domain structure of AvrPtoB. Numbers below the bar show points of cleavage with the protease subtilisin. The blue bar indicates the region sufficient for recognition by the plant Pto kinase for the mounting of PCD uninhibited by the CTD, whereas red bars indicate regions necessary or sufficient for anti-PCD activity in plants (7). (B) Overall structure of AvrPtoB(436–553): in blue, a “core fold” contributing most of the hydrophobic core and stabilizing the domain, in red, the long extended loops linking elements of secondary structure and which are critical to function (see text), in yellow, the extended, partially disordered N-terminal region that packs loosely against the core fold. Dotted lines indicate connecting regions of polypeptide not modeled because of disorder. N, N terminus; C, Cterminus. (C) Molecular surface of the AvrPtoB CTD aligned as in panel (B). The surface is colored by relative electrostatic potential, such that red is acidic (or negatively charged) and blue is basic (or positively charged).

Unexpectedly, the AvrPtoB CTD shows remarkable homology to the RING-finger and U-box families of proteins involved in ubiquitin ligase complexes in eukaryotes (Fig. 2A) (1016). This similarity begins in AvrPtoB at residue 476, extends to the C terminus of the protein, and encompasses the “core-fold” secondary structural elements of this ββαβ family (a three-stranded β sheet with a single helix and two extended loops). For example, alignments of AvrPtoB with the human Rbx1 RING-finger and the U-box domain of AtPUB14 from Arabidopsis have root-mean-square deviations in Cα positions of 2.4 Å (52 residues) and 1.1 Å (52 residues), respectively (10, 16, 17). We find the AvrPtoB CTD more similar to eukaryotic U-boxes, however, judging from both structural alignments and the lack of zinc coordination in the fold to support the extended loops (15, 16).

Fig. 2.

AvrPtoB mimics host RING-finger and U-box proteins. (A) Structural alignment of the AvrPtoB CTD (the core fold) with the RING-finger and U-box structures of Rbx1 [Protein Data Bank (PDB) ID 1LDJ, Rag1 (PDB ID 1RMD), PrP19 (PDB ID 1N87), and AtPUB14 (PDB ID 1T1H). (B) Visualization of the E2–binding site residues of Rbx1 with homologous regions in AvrPtoB and AtPUB14 [the polypeptide backbone from the alignment in panel (A)]. The corresponding residue numbers are indicated next to the amino acid. (C) Structure-based sequence alignment of AvrPtoB, Rbx1, and AtPUB14 focusing on the conserved, core fold. Secondary structure of AvrPtoB is indicated in blue above the sequence. Black highlights the three putative E2-binding residues shown in panel (B). The location of the AvrPtoB disordered insertion is indicated.

This similarity to eukaryotic RING-finger and U-box proteins extends beyond the conserved fold to crucial functional aspects of this family of proteins. In particular, a highly conserved binding site for recruiting elements of the ubiquitin ligase machinery is present in this bacterial virulence factor. The E3 ubiquitin ligases have a substrate (or adaptor-substrate) binding site, as well as a site to recruit proteins conjugated to ubiquitin so as to mediate transfer to the substrate—the so-called E2 “ubiquitin-conjugating” enzymes (18). These E2 enzymes are found to bind to a conserved region in eukaryotic RING-finger proteins (conserved in U-box proteins) that is characterized in part by a spatially clustered, three–amino acid surface patch (10, 16). This patch contains a highly conserved proline from the second large loop in this fold (following the first helix), a bulky hydrophobic residue in the first helix, and a hydrophobic residue in the first large loop. Mutation of these residues typically impairs E2 binding and ligase activity (10). The AvrPtoB CTD has a highly conserved patch very similar to those found in eukaryotic RING-finger and U-box proteins, and both the nature of the residues and their spatial positioning is highly conserved (Fig. 2, B and C). In particular, the proline is conserved (AvrPtoB Pro533), and large, hydrophobic residues (AvrPtoB Phe479 and Phe525) superimpose well on known E2 binding residues in eukaryotic RING-finger and U-box proteins (Fig. 2B).

This analysis of the structure raised the possibility that P. syringae AvrPtoB is functioning as a mimic of host E3 ubiquitin ligases. To test this hypothesis, we examined full-length and CTD AvrPtoB constructs for ubiquitin ligase activity and, in particular, for the common capacity seen in eukaryotic E3 ligases to autoubiquitinate. We performed ubiquitination assays (9) in vitro using exclusively recombinant, purified proteins (ubiquitin, E1 and E2 enzymes) in the presence of adenosine triphosphate (ATP), and full-length AvrPtoB, as well as the crystallized CTD construct fused to glutathione S-transferase (GST). Assays were performed with human UbcH5c, an E2 enzyme that showed activity in a screen of eight E2 enzymes (9), as well as a homologous Arabidopsis E2, AtUBC8. Similar results were obtained with both E2s: in the presence of full-length or CTD, E1, E2, ubiquitin, and ATP, a characteristic ladder to high molecular weight was observed in immunoblots with a monoclonal antibody to ubiquitin, indicating the addition of multiple ubiquitin molecules (Fig. 3A). A time course confirmed a gradual accumulation of higher molecular weight species of polyubiquitinated proteins (Fig. 3B). Probing the blot with an antibody against GST (to label the GST-AvrPtoB CTD fusion protein) or an antibody against His6 (to visualize the recombinant E2 enzymes tagged with a hexahistidine sequence) revealed that the banding is mostly due to autoubiquitination of AvrPtoB. The lack of a single component—E1, E2, ubiquitin, or the full-length or CTD AvrPtoB constructs—abolished this activity (Fig. 3A). We examined whether the putative E2 binding site identified in the structure was important for this ubiquitin ligase activity. Alanine substitutions were made in each of the three conserved E2–binding site residues of AvrPtoB (figs. S4 and S5) and examined in the assay described above. Each of the mutants lost ubiquitination activity, which indicated that the putative E2 binding site in AvrPtoB is critical to the ubiquitin ligase activity of the CTD (Fig. 3A). Together, these results strongly suggest that AvrPtoB, and in particular the CTD, is an active E3 ligase and likely mimics eukaryotic enzymes of this family in both structure and function.

Fig. 3.

The AvrPtoB CTD has eukaryotic-like ubiquitin ligase activity. (A) Autoubiquitination activity of AvrPtoB full-length protein (FL) and a CTD GST–AvrPtoB(436–553) fusion that was used for crystallization [labeled GST-(436–553]. In vitro ubiquitination assays were performed in the presence of ATP with the indicated combinations of proteins as described in (9). Proteins were resolved by SDS–polyacrylamide electrophoresis (SDS-PAGE) and were subjected to immunoblot analysis with indicated antibodies. Polyubiquitinated forms of AvrPtoB FL and AvrPtoB(436–553) were detected with antibodies against Ub or against Ub and GST, respectively. Antibodies against His6 were used for detection of 2XHis6-tagged ubiquitin-conjugating proteins (E2, human UbcH5c or its Arabidopsis homolog AtUBC8; colored in red; position indicated with arrows). FL mutants F479A, F525A, and P533A (33) are the E2–binding site mutants of full-length AvrPtoB. E1, ubiquitin-activating enzyme; Ub, ubiquitin. One asterisk indicates position of GST–AvrPtoB(436–553); two asterisks indicate the position of GST. SYPRO FL: gel was stained with SYPRO Ruby protein gel stain, and the position of wt FL AvrPtoB and each E2–binding site mutant is indicated by a black arrow. (B) Time-dependent in vitro autoubiquitination of GST–AvrPtoB(436–553). Reaction mix containing E1, E2 (Arabidopsis His6-UbcH8), GST–AvrPtoB(436–553), and ubiquitin was incubated for indicated times at 30°C as described (9), visualized by SDS-PAGE. The ubiquitinated proteins, GST–AvrPtoB(436–553), and His6-UbcH8 were detected in immunoblot analysis with the indicated antibodies as in Fig. 3A.

We next examined the importance of this E2 binding site in an infection assay in planta. There appear to be two distinct mechanisms that lead to AvrPtoB-mediated PCD. The first is dependent on both the Pto and Prf resistance (R) proteins in resistant tomato: AvrPtoB elicits PCD on the RG-PtoR tomato line that contains both R proteins, but does not elicit PCD on pto or prf mutant plants, RG-pto11 and RG-prf3, respectively (19). AvrPtoB is thus not able to suppress PCD triggered by the Pto R protein. The second mechanism by which AvrPtoB triggers PCD is Pto-independent and involves another putative R protein (Rsb), along with Prf. In plants lacking Pto-mediated resistance, AvrPtoB acts as a suppressor for PCD activated by the Rsb. Thus, loss of AvrPtoB anti-PCD activity (for example, through a deletion of the CTD, leaving residues 1 to 387, designated NTD) results in a gain of Rsb-mediated PCD on the RG-pto11 line (Fig. 4A). Rsb-mediated PCD in the RG-pto11 line therefore provides an assay to screen for anti-PCD activity of AvrPtoB by looking for a gain of PCD.

Fig. 4.

Mutations in E2-binding residues abolish anti-PCD activity and reduce virulence. (A) Loss of AvrPtoB anti-PCD activity in E2–binding residue mutants. PCD is observed as darkened regions on ethanol-cleared tomato leaves after infiltration by Agrobacterium strains carrying avrPtoB transgenes. Colored arrows correspond to the E2–binding site mutants (F479A, F525A, P533A; top panels), and wild-type AvrPtoB (WT), AvrPtoB from amino acids 1 to 387 (NTD), or empty vector control (bottom panels). The red circle depicts anti-PCD activity of wild-type AvrPtoB on RG-pto11, which is abolished in all E2–binding residue mutants and NTD. Schematic shows that AvrPtoB CTD inhibits Rsb-mediated, but not Pto-mediated, PCD. (B) Bacterial growth on tomato leaves inoculated with P. syringae pv. tomato DC3000::DavrPtoB expressing the WT AvrPtoB, NTD, or the E2–binding site mutants. The E2–binding site mutants elicit Pto-independent immunity on RG-pto11 leaves and consequently grow less than WT. All strains cause disease on susceptible RG-prf3 and elicit immunity on RG-PtoR. Growth was measured 6 days post inoculation as colony-forming units (CFU) per square centimeter of leaf area, and the experiment was repeated three times.

We began by examining the effects of transient expression of AvrPtoB constructs in tomato leaves (9). The three E2–binding site mutants that abolished E3 ligase activity elicited PCD on RG-PtoR and did not elicit PCD on RG-prf3, which revealed that these proteins are expressed and recognized by Pto and are not toxic to tomato (Fig. 4A). However, like the AvrPtoB NTD, and unlike the wild-type protein control, all three E2–binding site mutants elicited PCD on RG-pto11 leaves (Fig. 4A), which indicated that these proteins have lost anti-PCD activity. We then examined the importance of the E2 binding site in the context of a P. syringae infection of tomato. The same mutants were first transformed into the P. syringae DC3000::ΔavrPtoB mutant and expressed from the native hrp promoter (20). All the mutants caused disease on susceptible RG-prf3 tomato plants (Fig. 4B). However, on RG-pto11, the E2–binding site mutants elicited Pto-independent host immunity, comparable to that of the AvrPtoB NTD and the immunity observed on resistant RG-PtoR tomato plants (Fig. 4B). Wild-type AvrPtoB, however, caused disease on RG-pto11 mutants, because it suppresses Pto-independent PCD mediated by Rsb. Therefore, the disruption of these putative E2–binding site residues abrogates AvrPtoB anti-PCD and virulence activities in tomato. These mutagenesis results directly connect the loss of ubiquitin ligase activity observed in vitro with the loss of anti-PCD and virulence in tomato, which indicates that it is the E3 ligase activity of AvrPtoB that is critical for cell death suppression and for increased virulence of P. syringae.

The role of E3 ligases as inhibitors of PCD is well established (2125). Interestingly, some viral proteins act in host cells as E3 ligases, although the exact mechanisms of their action remain unclear (2629). Our data suggest that AvrPtoB functions as an E3 ligase in the infected cell, recruiting E2 enzymes and substrates (directly or in concert with another protein) and transferring ubiquitin or a ubiquitin-like molecule to cellular proteins involved in the regulation of PCD. An important strategy used by bacterial pathogens to manipulate host tissues is the functional mimicry of eukaryotic biochemical processes, and this mimicry often extends to the structural level (30). The use of eukaryotic ubiquitin-mediated systems for protein degradation by bacterial pathogens has been recently documented (31, 32). Salmonella temporally regulates bacterial invasion and host cell recovery through two T3SS substrates that have differing half-lives within host cells (31). In addition, the VirF protein of Agrobacterium contains an F-box motif that targets it to plant homologs of the E3 adaptor protein Skp1, which leads to degradation of the plant nuclear factor VIP1 (32). These bacterial factors differ from AvrPtoB, however, in that they rely on exploiting a host E3 ligase system, rather than embodying such enzymatic activity. Because AvrPtoB links E3 ubiquitin ligases and PCD and, furthermore, extends beyond plants to yeast cells as well as PCD activated in plants by the BAX protein, the bacterium has evolved this E3 ligase mimic to target a fundamental aspect of PCD that is highly conserved across several branches of eukaryota.

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Materials and Methods

Figs. S1 to S5

Table S1


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