Molecular basis for the RIN4 negative regulation of RPS2 disease resistance

Abstract

Recent studies have demonstrated that RPS2, a plasma membrane-localized nucleotide binding site/leucine-rich repeat protein from Arabidopsis thaliana, associates with RPM1 Interacting Protein 4 (RIN4) and that this association functions to modulate the RPS2-mediated defense pathway in response to the bacterial effector protein AvrRpt2. In addition to negatively regulating RPS2 activity, RIN4 is also a target of AvrRpt2, a Cys protease and cognate bacterial effector protein of RPS2. Nicotiana benthamiana has been employed as a heterologous expression system to characterize the RPS2-RIN4 association, defining the domains in RIN4 required for the negative regulation of RPS2 activity. Upon inoculation of N. benthamiana leaves with Agrobacterium tumefaciens expressing RPS2, a rapid hypersensitive response (HR) is detected with 22 h of infiltration. The HR can be blocked by infiltrating the leaf with A. tumefaciens expressing RPS2 in the presence of RIN4, recapitulating the ability of RIN4 to interfere with RPS2-mediated resistance in Arabidopsis. Moreover, in the presence of RIN4, the RPS2-mediated HR can be restored by the delivery of AvrRpt2 via A. tumefaciens. This assay has been developed as a phenotypic marker for (1) the HR-inducing phenotype associated with RPS2, (2) negative regulation of RPS2 by RIN4, and (3) the AvrRpt2-mediated disappearance of RIN4. Here, we present a series of deletion and site-directed mutation analyses to identify amino acids in RIN4 required for the RPS2-RIN4 association and to distinguish these from residues in RIN4 that serve as a target sequence for AvrRpt2. In addition to characterizing the RPS2-RIN4 association in N. benthamiana, we have moved forward to show that the biological relevance of these amino acid changes is applicable in Arabidopsis as well. To this end, we have identified specific amino acids within the C-terminal half of RIN4 that are required for RPS2 regulation and association.

Figure 1.

Coimmunoprecipitation of RPS2:HA and T7:RIN4 in N. benthamiana. (A) RIN4 negatively regulates RPS2 activation. N. benthamiana leaves were hand-infiltrated with Agrobacterium strains expressing either 35S:RPS2:HA (OD₆₀₀ = 0.1), 35S:T7:RIN4 (OD₆₀₀ = 0.4), or 35S:AvrRpt2:HA (OD₆₀₀ = 0.1). Immunoblot of HA- and T7-tagged proteins isolated 24 h postinoculation from wild-type N. benthamiana leaves hand-infiltrated with Agrobacterium strains expressing RPS2:HA, T7:RIN4, and AvrRpt2:HA confirmed expression of the various protein constructs (data not shown). Leaves were photographed 24 h postinoculation. (B) Protein gel blot analysis of protein extracts isolated from N. benthamiana leaves as diagramed in (A). Leaf punches (7 mm) were taken at 20 h, and protein samples were prepared as described in Methods. Immunoblots were probed with either α-HA (RPS2/AvrRpt2) or α-T7 (RIN4). Sizes are indicated at the left of each blot. (C) T7:RIN4 and RPS2:HA associate in planta. Immunoblots of immunoprecipitated proteins from the indicated tissue sources (center grid). “Input” lanes represent extracted samples incubated in the absence of antibody. “IP” indicates antibody used for immunoprecipitation and in all cases was both a positive and negative control for the fidelity of the antibody. “co-IP” refers to the coimmunoprecipitation experiment designed to detect the T7:RIN4 and RPS2:HA association in planta. Top panel, HA-HRP immunoblot; bottom panel, T7-HRP immunoblot. Protein sizes are indicated to the left of each blot.

Figure 2.

Strategy for Identifying the Domain(s) of RIN4 Necessary for the Negative Regulation of RPS2 Function. Using a PCR-based approach, deletion constructs were made in RIN4 corresponding to stepwise deletions, resulting in the elimination of amino acids at both the C and N termini of RIN4. Abrogation (+) refers to RIN4 ability to block RPS2 activation. Cleavage (+) refers to AvrRpt2 ability to cleave wild-type RIN4 at either the N- or C-terminal cleavage site (RCS1 or RCS2, respectively) or both and, where indicated, the various RIN4 deletion/mutant constructs. Where designated, “NA” refers to not applicable, indicating the respective cleavage site is absent in the construct. HR (+) refers to induction of the RPS2-mediated HR after delivery of AvrRpt2. Key amino acid residues are shown when necessary. Inverted triangles designate the AvrRpt2 cleavage sites present in both the N and C termini of RIN4.

Figure 3.

Negative Regulation of RPS2 by RIN4: The Negative Regulatory Activity of RIN4 Is Localized in the C Terminus. RPS2:HA and T7:RIN4 C and N termini constructs were transiently expressed in wild-type N. benthamiana plants via Agrobacterium-mediated transformation. (A) Inoculation of N. benthamiana leaves with Agrobacterium expressing RPS2:HA resulted in the rapid (∼18 h) induction of the HR (first leaf panel, top). Leaf segments were photographed 24 h postinoculation. Coexpression of RPS2:HA and T7:RIN4-C resulted in a complete inhibition of the HR (second leaf panel, bottom). Conversely, coinoculation of N. benthamiana leaves with RPS2:HA and T7:RIN4-N failed to inhibit the induction of the HR (fourth leaf panel, top). Control inoculations with Agrobacterium expressing AvrRpt2 (second leaf panel, top) and the T7:RIN4 termini alone (i.e., RIN4-N and RIN4-C) showed only a slight HR phenotype encircling the periphery of the inoculation point or no reaction at all, respectively. (B) The N terminus of RIN4 (i.e., T7:RIN4-N) does not coimmunoprecipitate RPS2:HA. Immunoblot of αHA and αT7 immunoprecipitated proteins isolated 24 h postinoculation from wild-type N. benthamiana leaves hand-infiltrated with Agrobacterium strains expressing RPS2:HA and the N-terminal half of RIN4 (i.e., T7:RIN4-N). Total protein extracts were immunoprecipitated with anti-HA (RPS2) and anti-T7 (RIN4) antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti-T7 (top panel) and anti-HA (bottom panel) antibodies. (C) The C terminus of RIN4 (i.e., T7:RIN4-C) coimmunoprecipitates RPS2:HA.

Figure 4.

Amino Acid Residues Necessary for Cleavage of RIN4 by AvrRpt2 and Negative Regulation of RPS2 by RIN4 Are Distinct and Differentially Localized. (A) Differential cleavage of transiently coexpressed cleavage site mutants of RIN4 in the presence of AvrRpt2. N. benthamiana leaves were coinfiltrated with Agrobacterium expressing AvrRpt2:HA (OD₆₀₀ = 0.1) and various cleavage site mutants of T7:RIN4 (OD₆₀₀ = 0.4). Twenty-four hours after coinfiltration, 7-mm leaf punches were harvested, ground in 3× Laemmli buffer, and analyzed by SDS-PAGE, followed by protein gel blotting with anti-RIN4 antibodies. Protein molecular mass sizes are shown at the left of the blot. The N-terminal T7-epitope tag on RIN4 was used to identify the N-terminal cleavage product released by coexpression with AvrRpt2. Parallel, duplicate samples were harvested at 24 h, homogenized in immunoprecipitation buffer (see Methods), and processed according to standard immunoprecipitation protocols as described in this study. Detection of the cleaved ∼1-kD T7-tagged N terminus was achieved by protein gel blot analysis using α-T7-HRP conjugated antibodies. The absence of a detectable RIN4 cleavage product in K150A/F151A + AvrRpt2 likely represents a lack of protein stability after cleavage at the C-terminal cleavage site. (B) All cleavage site mutants retain their ability to negative regulate RPS2 activation. Top row: leaf segments in the absence of AvrRpt2 coinfiltration. Note that all cleavage site mutants of RIN4 retain the ability to block the RPS2-induced HR. Bottom row: leaf phenotypes in the presence of AvrRpt2 coinfiltration. RPS2 and AvrRpt2 control inoculations are shown as indicated. Leaf segments were photographed 24 h postinoculation.

Figure 5.

The C-Terminal Amino Acids in RIN4 Are Required for Regulating RPS2 Activation. Stepwise deletions of two, six, and nine amino acids from the C terminus of RIN4, as well as a single amino acid change (A180P and C203R) results in a gradual (stepwise) or complete (A180P and C203R) loss in the regulatory function of RIN4 activity. (A) N. benthamiana leaves were coinfiltrated with Agrobacterium expressing AvrRpt2:HA (OD₆₀₀ = 0.1) and various C-terminal deletion mutants of T7:RIN4 (OD₆₀₀ = 0.4). Top row: constructs infiltrated in the absence of Agrobacterium:AvrRpt2:HA. Bottom row: C-terminal deletion constructs coinfiltrated in the presence of AvrRpt2:HA. Control infiltrations (RPS2:HA and AvrRpt2:HA) are shown as indicated. All leaf segments were photographed 24 h postinoculation. (B) Protein gel blot analysis of deletion and single site mutants alone and in the presence of AvrRpt2:HA. (C) Coimmunoprecipitation: T7:RIN4_C203R does not associate with RPS2:HA in planta.

Figure 6.

Transient Overexpression of RIN4 in ED-Inducible RPS2:HA Arabidopsis Plants Blocks Induction of the RPS2-Mediated HR. Recapitulation of abrogation/regulation experiments from N. benthamiana transient assays in Arabidopsis (rps2/pER8:RPS2:HA). (A) Addition of 20 μM ED induces expression of RPS2:HA, followed by subsequent activation of the HR leading to cell death (first leaf panel). Coinoculation with various Agrobacterium-RIN4 clones results in the complete block of RPS2 activation (+20 μM ED + T7:RIN4) to a loss of negative regulation (second to fourth panels). Coinfiltration of ED in the presence of Agrobacterium expressing green fluorescent protein mimics RPS2 induction in the absence of RIN4 (control, fifth panel). Inoculation of rps2/pER8 vector only control plants with 20 μM ED showed no response (i.e., no HR; data not shown). (B) Top row: induction (first panel) and abrogation (second panel) of the RPS2-mediated HR via transient expression in Arabidopsis by wild-type RIN4. Middle row: delivery of AvrRpt2 via the TTSS of Pseudomonas syringae pv tomato (Pst) results in the elimination of RIN4, release of the negative regulation of RPS2, and subsequent restoration of the HR (first panel). Bottom row: coinoculation of 20 μM ED and Pst DC3000 (–AvrRpt2) shows the onset of disease symptoms, evidenced by an absence of both pathogen recognition and the loss in induction of the RPS2-mediated HR. Results of cleavage site mutants are shown as indicated. Corresponding protein gel blot analysis is shown in Supplemental Figure 3 online.