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. 2014 Jul 24;10(7):e1004227.
doi: 10.1371/journal.ppat.1004227. eCollection 2014 Jul.

Pto kinase binds two domains of AvrPtoB and its proximity to the effector E3 ligase determines if it evades degradation and activates plant immunity

Johannes Mathieu  1 , Simon Schwizer  2 , Gregory B Martin  2
Affiliations

Pto kinase binds two domains of AvrPtoB and its proximity to the effector E3 ligase determines if it evades degradation and activates plant immunity

Johannes Mathieu et al. PLoS Pathog. .

Abstract

The tomato--Pseudomonas syringae pv. tomato (Pst)--pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pst virulence protein AvrPtoB and activate effector-triggered immunity (ETI). AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450) in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pto, but not Fen, binds the PID of AvrPtoB and is recalcitrant to E3 ligase-mediated degradation.
(A) Schematic of AvrPtoB functional domains and truncations used in this and previous publications . PID, Pto-interacting domain ; FID, Fen-interacting domain (also known as the Bak1-interacting domain [25]). Position of amino acid substitutions, F173A and G325A, are shown. (B) Yeast two-hybrid analyses testing the interaction of tomato Pto and Fen kinases (in the bait vector) with different functional domains in AvrPtoB using truncated forms of the effector (in the prey vector). Blue patches indicate a positive interaction. Pto interacted with both the PID and the FID, whereas Fen exclusively bound the FID. wt, wild-type AvrPtoB in the truncation indicated; EV, empty vector. (C) Yeast two-hybrid analyses of the binding properties of tomato Pto and Fen kinases (in the bait vector) towards full-length AvrPtoB protein (in the prey vector). Blue patches indicate a positive interaction. Pto bound to the FID, proximal to the E3 ubiquitin ligase domain, was degraded in an E3-ligase dependent manner, similar to Fen. E3-LOF, AvrPtoB(F479A/F525A/P533A) has substitutions that abolish binding of the E2 conjugating enzyme and lacks E3 ligase activity ; EV, empty vector.
Figure 2
Figure 2. AvrPtoB mediates Pto degradation if the kinase is positioned closer to the AvrPtoB E3 ligase.
(A) The AvrPtoB PID was fused to the E3 ligase domain, cloned into the prey vector (see Methods for details) and tested for its interaction with Pto (in the bait vector). Blue patches indicate a positive interaction. Pto was degraded if it was in closer proximity to the E3 ligase domain as shown by the white patches. E3-LOF, AvrPtoB(F479A/F525A/P533A) lacks E3 ligase activity; EV, empty vector. (B) Expression levels of both fusion proteins and wild-type AvrPtoB are similar. Yeast cells grown in inductive medium were harvested, normalized by OD600, boiled in Laemmli buffer and proteins resolved by Western blotting. Fusion proteins were detected using ant-HA primary (clone 3F10, Roche, Indianapolis, IN, USA) and anti-rat-800 secondary (IRDye 800CW, LI-COR, Lincoln, NE, USA) antibodies and visualized using an Odyssey Scanner (LI-COR). CBB, Coomassie brilliant blue staining.
Figure 3
Figure 3. Pto binds to the FID in plant cells and is degraded by activity of the AvrPtoB E3 ligase.
(A) The P. syringae pv. tomato strain DC3000ΔavrPtoΔavrPtoB was transformed with wild-type (wt) AvrPtoB or AvrPtoB variants under control of an Pst hrp promoter and infiltrated into leaves of Rio Grande (RG) tomatoes. Disease phenotypes were consistent with the observations in yeast two-hybrid analyses. RG-PtoR (Pto/Pto, Prf/Prf); RG-prf3 (Pto/Pto, prf3/prf3). RG-prf3 has a deletion in Prf that inactivates the Pto/Prf pathway. (B) Tomato Pto and Prf were co-expressed with different variants of AvrPtoB in a leaf of Nicotiana benthamiana by using Agrobacterium-mediated transient transformation. Pto activated ETI, as manifested by cell death with AvrPtoB(F173A)-E3-LOF, upon binding the FID but this response was repressed by the E3 ligase domain with no cell death visible with AvrPtoB(F173A), similar to Fen binding of the same domain. E3-LOF, AvrPtoB(F479A/F525A/P533A) lacks E3 ligase activity; YFP, yellow fluorescent protein control. (C) Abundance of Pto is decreased when it is co-expressed with AvrPtoB variants having an active E3 ligase domain. Using Agrobacterium-mediated transient transformation, tomato Pto was co-expressed with different variants of AvrPtoB in a leaf of a Prf-silenced Nicotiana benthamiana plant (to avoid the possible effect of cell death on protein abundance). Leaf samples were harvest 48 hrs after agroinfiltration and proteins were isolated for Western blot analysis. Similar experiments were done with yellow fluorescent protein (YFP) as a loading control. Proteins were detected using an anti-c-Myc-HRP antibody targeted to the epitope tag fused to each protein. Exposure was 1 min for AvrPtoB and Pto, and 10 seconds for YFP. CBB, Coomassie brilliant blue staining.
Figure 4
Figure 4. Pto-mediated phosphorylation of T450 in AvrPtoB does not impact Pto recalcitrance to E3 ligase-mediated degradation.
(A) Yeast two-hybrid analyses of the interaction of AvrPtoB(T450A) and AvrPtoB(T450D) with Pto and Fen. AvrPtoB or the variants shown (in the prey vector) were tested for their interaction with Pto and Fen. Blue patches indicate a positive interaction. Substitution of T450 in AvrPtoB by the non-phosphorylatable residue alanine had no impact on either the capability of the effector to degrade Fen or the recalcitrance of Pto to this degradation. AvrPtoB(T450D) interacted with Pto as expected based on results in (B) and (C) below. The reason for the lack of Fen interaction with AvrPtoB(T450D) is unknown as this interaction would be expected to occur based on (B) and evidently does occur in the plant-pathogen interaction as shown in (C). E3-LOF, AvrPtoB(F479A/F525A/P533A) lacks E3 ligase activity; EV, empty vector. (B) In vitro ubiquitination assay to determine ubiquitin ligase activities of different variants of AvrPtoB. GST-fusions of the effector were purified from E. coli and subjected to an in vitro ubiquitination assay. After Western blotting, poly-ubiquitin chains were detected using a monoclonal anti-ubiquitin antibody (α-Ub). In contrast to previous reports, AvrPtoB(T450A) retained significant E3 ubiquitin ligase activity. CBB, Coomassie Brilliant Blue. Black divider line in loading control panel demarcates a copy/paste border as GST has a much smaller mass than the fusion proteins. (C) The P. syringae pv. tomato strain DC3000ΔavrPtoΔavrPtoB was transformed with wild-type (wt) or variants of AvrPtoB under control of a Pst hrp promoter and infiltrated into Rio Grande (RG) tomatoes. AvrPtoB(T450D) was recognized both by Pto (no disease in RG-PtoR) and Fen (no disease in the absence of Pto in RG-pto11) and mimics the E3-LOF version of the effector. In contrast, AvrPtoB(T450A) behaved identical to wild-type AvrPtoB. Fen-mediated immunity is suppressed, but Pto can still resist degradation and confer immunity, demonstrating that phosphorylation of T450 in AvrPtoB is not required for Pto to resist degradation in vivo thus confirming the in vitro finding that AvrPtoB(T450A) has E3 ligase activity. RG-PtoR, Pto/Pto, Prf/Prf; RG-pto11, pto11/pto11, Prf/Prf; RG-prf3, Pto/Pto, prf3/prf3. RG-pto11 has a point mutation that produces a non-functional Pto protein. RG-prf3 has a deletion in Prf.
Figure 5
Figure 5. Fen has higher kinase activity than Pto and Pto(G50S) has little or no kinase activity.
(A) In vitro kinase assay for tomato Pto, Fen, and Pto(G50S) at pH6.8. At this pH, no brown discoloration was visible upon addition of 10 mM MnCl2, indicating a better availability of Mn2+. Kinase buffers were supplemented with 10 mM MnCl2, 10 mM MgCl2 or 10 mM of each. Under these conditions, Fen showed a higher kinase activity than Pto and Pto(G50S) had little or no kinase activity. Coomassie Brilliant Blue (CBB) staining showed similar amounts of the kinases were present. (B) Phosphoprotein-specific ProQ staining of Pto, Fen and Pto(G50S) to assess their phosphorylation status in bacteria. Pto and Fen were expressed in E. coli, pulled down using MBP-agarose, resolved by SDS-PAGE and subjected to ProQ staining. Stronger staining of Fen indicates a higher autophosphorylation activity in situ. Pto(G50S) had little or no kinase activity in this in vivo assay. Coomassie Brilliant Blue (CBB) staining showed similar amounts of the kinases were present.
Figure 6
Figure 6. Pto and Fen kinase activities are dispensable for activation of AvrPtoB-elicited ETI.
The Pto(G50S) variant which has little or no kinase activity (see Fig. 5BC) and tomato Prf were co-expressed with different variants of AvrPtoB in a leaf of Nicotiana benthamiana by Agrobacterium-mediated transient transformation. The response of Pto(G50S) to the AvrPtoB variants was indistinguishable from wild-type Pto in that it caused ETI-associated cell death upon binding the FID (cell deaths with AvrPtoB(F173A; E3-LOF)), but this response was repressed by the E3 ligase domain (no cell death by AvrPtoB(F173A)).
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