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Comparative Study
, 133 (3), 1072-82

Cleavage of the Pseudomonas Syringae Type III Effector AvrRpt2 Requires a Host Factor(s) Common Among Eukaryotes and Is Important for AvrRpt2 Localization in the Host Cell

Affiliations
Comparative Study

Cleavage of the Pseudomonas Syringae Type III Effector AvrRpt2 Requires a Host Factor(s) Common Among Eukaryotes and Is Important for AvrRpt2 Localization in the Host Cell

Ping Jin et al. Plant Physiol.

Abstract

Many phytopathogenic bacteria use a type III secretion system to deliver type III effector proteins into the host plant cell. The Pseudomonas syringae type III effector AvrRpt2 is cleaved at a specific site when translocated into the host cell. In this study, we first demonstrate that the factor(s) required for AvrRpt2 cleavage is present in extracts from animal and yeast cells, as well as plant cells. The cleavage factor in animal and plant cell extracts was heat labile but relatively insensitive to protease inhibitors. Second, mutational analysis of AvrRpt2 was applied to identify features important for its cleavage. In addition to two of the amino acid residues in the immediate vicinity of the cleavage site, a large part of the region C-terminal to the cleavage site was required when AvrRpt2 was cleaved in animal cell extract. Most of these features were also important when AvrRpt2 was cleaved in plant cells. Third, we investigated the effect of cleavage in interactions of AvrRpt2 with plant cells. Cleavage of AvrRpt2 appeared to be important for proper interactions with Arabidopsis cells that lack the resistance gene product corresponding to AvrRpt2, RPS2. In addition, removal of the region N-terminal to the cleavage site was important for the correct localization of the C-terminal effector region of the protein in the host cell. We speculate that the virulence function of AvrRpt2 requires removal of the N-terminal region to redirect the effector protein to a specific subcellular location in the host cell after translocation of the protein.

Figures

Figure 1.
Figure 1.
AvrRpt2 can be cleaved in rabbit reticulocyte lysate. a, Smaller products observed are cleaved products of AvrRpt2. AvrRpt2 (lanes 1–3), GST::AvrRpt2 (lanes 4–8), and AvrB (lanes 9–11) were translated and 35S-labeled using rabbit reticulocyte lysate. The durations of incubations are indicated. In lanes 7 and 8, EDTA was added to a final concentration of 20 mm after a 0.5-h in vitro translation reaction to stop the reaction. The hours indicated are the total of the reaction time and the incubation time following the EDTA addition. The labeled proteins were resolved by denaturing PAGE and were detected by phosphor imaging of the gel. b, AvrRpt2 is cleaved at the correct site in rabbit reticulocyte lysate. The cleaved products of the GST::AvrRpt2 reaction (lane 2) were compared with GST::ΔC72 (lane 1), which corresponds to the N-terminal region expected if the cleavage occurs between G71 and G72, and with ΔN71 (lane 3), which corresponds to the expected C-terminal region. White triangle, Full-length; black triangle, C-terminal region after cleavage; white circle, N-terminal region after cleavage. The N-terminal region was not observed with AvrRpt2 (a, lanes 1–3) due to its small size. The positions of molecular markers are indicated on the right.
Figure 2.
Figure 2.
The cleavage factor(s) is prevalent among eukaryotes. a, E. coli-produced AvrRpt2::his is properly cleaved with rabbit reticulocyte lysate, Arabidopsis extracts, and yeast extracts. AvrRpt2::his was produced in E. coli and purified by affinity chromatography. The purified protein was incubated with buffer control (lanes 1 and 7), reticulocyte lysate (lanes 2 and 3), Arabidopsis extracts (lanes 4 and 5), or yeast extracts (lane 8) for the indicated time (2.5 h for lanes 7–9). Then the reactions were resolved by denaturing PAGE, and the gel was subjected to immunoblot analysis using anti-(his)3 antibody. ΔN71::his (lanes 6 and 9) was included as a molecular marker for the post-cleavage C terminus. The left and right panels represent separate experiments. b, The cleavage factors in rabbit reticulocyte lysate and in Arabidopsis extracts are heat labile and partially sensitive to protease inhibitors. Reticulocyte lysate (lanes 1–4) and Arabidopsis extracts (lanes 5–8) were treated with either heat (5 min in boiling water, lanes 1,2, 5, and 6) or a cocktail of protease inhibitors (lanes 3, 4, 7, and 8). E. coli-produced AvrRpt2::his was incubated with the treated extracts for the indicated time. The reactions were analyzed by immuno-blot as described in a. c, A bovine actin fraction contains a weak cleavage activity. AvrRpt2::his was incubated with an actin fraction for 1 h (lane 2) and was analyzed by immunoblot as in a. The full-length protein (lane 1) and the expected C-terminal region after cleavage (lane 3) were included as positional markers. The efficiency of cleavage can be compared by the ratio between the full-length and cleaved forms in each lane. White triangle, Full-length; black triangle, C-terminal region.
Figure 3.
Figure 3.
A large part of the C-terminal region of AvrRpt2 is required for cleavage. The AvrRpt2 derivatives indicated were translated and 35S-labeled in rabbit reticulocyte lysate. The reactions were incubated for the number of hours indicated. The reactions were analyzed as in Figure 1. The derivatives were made in either the GST::AvrRpt2 context (lanes 1–18) or the GST::ΔN56 context (lanes 19–24). White triangle, Full-length; black triangle, C-terminal region; white circle, N-terminal region.
Figure 4.
Figure 4.
Cleavage of AvrRpt2 derivatives in the plant cell. The indicated AvrRpt2::GFPf derivatives were expressed in Arabidopsis rps2 mutant protoplasts. The extracts from the protoplasts were analyzed by immunoblot as in Figure 2 except that the anti-FLAG antibody was used. AvrRpt2::GFPf was included in every separate experiment as a positive control. a through c represent results from three different experiments. White triangle, Full-length; black triangle, C-terminal region.
Figure 5.
Figure 5.
RPS2 recognition of AvrRpt2 derivatives. The indicated AvrRpt2::f derivatives were tested for recognition by RPS2 by a transient expression assay either with (black bars) or without (gray bars) RPS2. When a derivative is active, the relative rLUC activity with RPS2 is significantly lower than it is without RPS2. The mean rLUC activity value for vector with RPS2 was set to 100 to normalize the entire results. Error bar = sd. The number of replicates, n = 4, except for vector with RPS2 (n = 40), AvrRpt2::f with RPS2 (n = 40), vector without RPS2 (n = 36), and AvrRpt2::f without RPS2 (n = 36).
Figure 6.
Figure 6.
AvrRpt2myr::GFPf is largely localized to the plasma membrane in the plant cell. GFP::f (a), AvrRpt2::GFPf (b), AvrRpt2myr::GFPf (c), and AvrB::GFP (d) were expressed in Arabidopsis protoplasts, and the GFP fluorescence was observed under a laser confocal microscope. In each panel, a light image is shown on the left, followed by six confocal optical section images from the bottom to the middle of the protoplast. Protoplasts exhibiting representative localization of the GFP-tagged proteins were chosen. Note that not all protoplasts express the derivatives. Nuclear staining is indicated by white arrowheads.
Figure 7.
Figure 7.
Cleavage of the N-terminal region of AvrRpt2 is important for subcellular localization in the host cell. GFP::f (a), AvrRpt2::GFPf (b), C122Y::GFPf (c), Δ67–76::GFPf (d), and F70A::GFPf (e) were expressed in Arabidopsis protoplasts, and the GFP fluorescence (green, on the left in each panel) and chlorophyll fluorescence (red, on the right in each panel) were observed under a laser confocal microscope. Chlorophyll fluorescence images indicate the positions of chloroplasts. The composite images of all the sections are shown. A representative fluorescence pattern for each derivative is shown. Nuclear staining is indicated by white arrowheads.
Figure 8.
Figure 8.
Type III effector redirection model. This model illustrates that cleaving off the N-terminal region, which contains the type III secretion signal, could redirect the type III effector to a particular subcellular location in the host plant cell. Many type III secretion signal sequences are similar to plastid-targeting signals, so an uncleaved type III effector could be transported into the plastid (If not cleaved). However, cleavage removes the cryptic plastid-targeting signal and exposes a new N terminus (Cleaved). If post-cleavage N terminus does not have a particular targeting signal, the C-terminal region stays in the cytosol, like AvrRpt2. If it has a targeting signal, the C-terminal region can be transported to an appropriate subcellular location. For example, AvrPphB is targeted to the plasma membrane via myristoylation of the new N terminus. PM, Plasma membrane; ER, endoplasmic reticulum.

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