The Pseudomonas syringae effector AvrRpt2 cleaves its C-terminally acylated target, RIN4, from Arabidopsis membranes to block RPM1 activation

Abstract

Plant pathogenic Pseudomonas syringae deliver type III effector proteins into the host cell, where they function to manipulate host defense and metabolism to benefit the extracellular bacterial colony. The activity of these virulence factors can be monitored by plant disease resistance proteins deployed to "guard" the targeted host proteins. The Arabidopsis RIN4 protein is targeted by three different type III effectors. Specific manipulation of RIN4 by each of them leads to activation of either the RPM1 or RPS2 disease resistance proteins. The type III effector AvrRpt2 is a cysteine protease that is autoprocessed inside the host cell where it activates RPS2 by causing RIN4 disappearance. RIN4 contains two sites related to the AvrRpt2 cleavage site (RCS1 and RCS2). We demonstrate that AvrRpt2-dependent cleavage of RIN4 at RCS2 is functionally critical in vivo. This event leads to proteasome-mediated elimination of all but a membrane-embedded approximately 6.4-kDa C-terminal fragment of RIN4. One or more of three consecutive cysteines in this C-terminal fragment are required for RIN4 localization; these are likely to be palmitoylation and/or prenylation sites. AvrRpt2-dependent cleavage at RCS2, and release of the remainder of RIN4 from the membrane, consequently prevents RPM1 activation by AvrRpm1 or AvrB. RCS2 is contained within the smallest tested fragment of RIN4 that binds AvrB in vitro. Thus, at least two bacterial virulence factors target the same domain of RIN4, a approximately 30-aa plant-specific signature sequence found in a small Arabidopsis protein family that may be additional targets for these bacterial virulence factors.

Fig. 1.

RIN4 contains two cysteine protease cleavage sites. (A) A consensus cysteine protease cleavage site is present in both RIN4 and AvrRpt2. Block mutations to alanine discussed below were made in the underlined residues, V-FG-W. (B) RIN4 amino acid sequence features: RCS1 (V6-W12), RCS2 (V148-W154), and a putative palmitoylation site at C203-C205 are underlined. The fragment from P142 to G179 (end points underlined) is discussed in the Fig. 6 legend. (C) Overexpression of proteolytically active AvrRpt2 leads to RIN4 cleavage and accumulation of a C-terminal RIN4 fragment. Col-0 transgenic plants conditionally overexpressing AvrRpt2 were treated with 20 μM DEX, and total extracts were prepared from leaves harvested at the indicated time points indicated (hours post-DEX) for SDS/PAGE and Western blot with anti-RIN4 sera. (D) Delivery of enzymatically active AvrRpt2 from P. syringae specifically results in accumulation of a 6.4-kDa membrane-bound RIN4 fragment, consistent with cleavage at RCS2. Col-0 plants were infected with 5 × 10⁷ colony-forming units/ml of Pto DC3000 carrying either avrRpt2 or a protease dead mutant avrRpt2C122A. Protein samples from the membrane microsomal fraction were tested by protein blot with anti-RIN4 sera at 0, 4, and 8 h postinfection.

Fig. 2.

RIN4 mutant derivative constructs used to test the effect of RIN4 proteolysis on RPM1 activation and AvrRpt2 virulence function. Two sets of conditionally expressed constructs to express mutant RIN4 proteins are shown. (A) The 211-aa RIN4 protein is schematically shown at the top. Putative AvrRpt2 cleavage sites follow G10 and G152. Putative palmitoylation sites at C203, C204, and C205 are denoted by a gray box. RIN4 derivatives that mimic the putative products of AvrRpt2-mediated cleavage were constructed: the larger product from N-terminal cleavage, termed RIN4_11-211, the product ending at the C-terminal cleavage site, RIN4_1-152, or the internal product of both cleavage events, RIN4_11-152. (B) Alanine RIN4 block mutants that alter AvrRpt2-mediated cleavage events at either the N-terminal cleavage site, termed RIN4_RCS1, the C-terminal cleavage site, RIN4_RCS2, or both, RIN4_RCS1-2 (see Fig. 1 for consensus sequences mutated to alanine). (C) Alanine block mutation (gray X) of the RIN4 putative palmitoylation site, C203A/C204A/C205A, generates RIN4_{C203AC204AC205A}.

Fig. 3.

RIN4 is localized to the membrane by a putative palmitoylation site at C203/C204/C205. (A) Membrane localization requires the RIN4 C-terminal region. The RIN4 derivatives that mimic AvrRpt2 cleavage (Fig. 2), RIN4_11-211, RIN4_1-152, and RIN4_11-152, were conditionally expressed in transgenic rin4 rps2 RPM1 plants (20 μM DEX treatment) for 24 h. Protein samples from the microsomal and soluble fractions were analyzed for presence of the respective RIN4 fragments. (B and C) The three possible cysteine palmitoylation sites, C203, C204, and C205 are required for RIN4 localization membrane. Conditionally expressed transgenes encoding WT RIN4 or the RIN4_{C203AC204AC205A} block mutant (Fig. 2) were constructed in rin4 rps2 RPM1 plants. Two transgenic lines for each construct were tested for localization of RIN4 either in the absence (B) or presence (C) of 20 μM clasto-lactacysteine β-lactone (see Materials and Methods). (D) A palmitoylation inhibitor diminishes the amount of RIN4 targeted to the membrane. 2-BPA (100 μM) was infiltrated 1 h before DEX treatment of transgenic rin4 rps2 RPM1 plants conditionally expressing WT RIN4, and samples were taken 24 h later for protein blots.

Fig. 4.

In vivo cleavage site of RIN4 by AvrRpt2 at RCS2 is critical. (A) Mutation of the AvrRpt2 cleavage sites in RIN4 does not alter their proper localization to the membrane. Membrane and soluble protein fractions were prepared from leaves 24 h after 20 μM DEX treatment of transgenic rin4 rps2 RPM1 plants conditionally expressing the proteins listed at the top (see Fig. 2). (B) AvrRpt2-dependent cleavage at RIN4_RCS2 is required for elimination of RIN4 from the membrane. Plants were treated with DEX as in A and inoculated 24 h later with 5 × 10⁵ colony-forming units/ml of Pto DC3000(EV), Pto DC3000(avrRpt2), or Pto DC3000 (avrRpt2C122A). Leaf samples were collected for microsomal membrane protein extraction 12 h after infection.

Fig. 5.

AvrRpt2-dependent cleavage at RIN4_RCS2 blocks activation of RPM1 by AvrRpm1. Plants expressing RIN4 and RCS mutant derivatives listed at the bottom were treated with DEX (as in Fig. 4) to induce expression of WT RIN4 and RCS mutant derivatives listed below and then inoculated with 5 × 10⁵ colony-forming units/ml of Pto DC3000-expressing genes listed. Bacterial growth was measured 0 (empty bars) and 3 days (filled bars) postinoculation. Error bars represent standard deviation from three samples. The values from the experiment are representative of two additional replicates.

Fig. 6.

The RIN4 domain that interacts with AvrB overlaps RCS2. (A) RIN4 deletion mutants were constructed and expressed in E. coli (see Materials and Methods) to define a region sufficient for interaction with AvrB in vitro. The location of RCS1 and RCS2 are indicated by boxes on the RIN4 sequence. (B) Purified AvrB and RIN4 deletion derivatives were incubated together before being run on native 12.5% PAGE gels. Addition of either RIN4_142-211 or RIN4_142-179 fragments with AvrB resulted in slower band migration (indicated by ^*). (C) Confirmation of the RIN4_142-179 fragment as the AvrB interacting domain was made by native gel filtration and subsequent SDS/PAGE of the relevant fractions. St, starting mixture of AvrB and RIN4_142-179. No other fractions contained either protein.