NDR1 interaction with RIN4 mediates the differential activation of multiple disease resistance pathways in Arabidopsis

Abstract

Recognition of pathogens by plants involves the coordinated efforts of molecular chaperones, disease resistance (R) proteins, and components of disease resistance signaling pathways. Characterization of events associated with pathogen perception in Arabidopsis thaliana has advanced understanding of molecular genetic mechanisms associated with disease resistance and protein interactions critical for the activation of resistance signaling. Regulation of R protein-mediated signaling in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis involves the physical association of at least two R proteins with the negative regulator RPM1 INTERACTING PROTEIN4 (RIN4). While the RIN4-RPS2 (for RESISTANCE TO P. SYRINGAE2) and RIN4-RPM1 (for RESISTANCE TO P. SYRINGAE PV MACULICOLA1) signaling pathways exhibit differential mechanisms of activation in terms of effector action, the requirement for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is shared. Using a yeast two-hybrid screen, followed by a series of coimmunoprecipitation experiments, we demonstrate that the RIN4-NDR1 interaction occurs on the cytoplasmically localized N-terminal portion of NDR1 and that this interaction is required for the activation of resistance signaling following infection by P. syringae expressing the Cys protease Type III effector protein AvrRpt2. We demonstrate that like RPS2 and RPM1, NDR1 also associates with RIN4 in planta. We suggest that this interaction serves to further regulate activation of disease resistance signaling following recognition of P. syringae DC3000-AvrRpt2 by Arabidopsis.

Figure 1.

Identification of the NDR1 and RIN4 Interaction by a Yeast Two-Hybrid Screen. A specific interaction between NDR1 and RIN4 was identified using the CytoTrap two-hybrid system. Approximately 10⁶ S. cerevisiae cdc25H cells comprising an Arabidopsis cDNA library made from pathogen and non-pathogen-treated tissues were screened using RIN4 (pSosRIN4) as a bait. Recapitulation of the interaction was performed on galactose-containing minimal media in the absence of uracil and leucine at 37°C (top panel). Control interactions, consisting of pSosMAFB+pMyrMAFBα (center panel, positive control) and pSOSMAFB+pMyrLamin-C (bottom panel, negative control) confirmed the NDR1–RIN4 interaction as specific.

Figure 2.

RIN4 and NDR1 Interact in Planta. Coimmunoprecipitation of RIN4 and NDR1 in N. benthamiana and Arabidopsis. (A) Transient coexpression in wild-type N. benthamiana plants. Immunoblot of anti-HA and anti-T7 immunoprecipitated proteins isolated 40 h after inoculation from wild-type N. benthamiana leaves hand-infiltrated with Agrobacterium strains expressing HA:NDR1 and T7:RIN4. Total protein extracts were immunoprecipitated with anti-HA (NDR1) and anti-T7 (RIN4) antibodies. Immunoprecipitated proteins were detected by immunoblotting anti-T7 (top panel) and anti-HA (bottom panel) immunoblots. Protein sizes are indicated at the left side of the immunoblots. (B) Expression and coimmunoprecipitation of HA:NDR1 and native RIN4 in Arabidopsis. Total protein extracts were immunoprecipitated with either anti-HA (positive control) or anti-RIN4 antibodies, followed by protein gel blot analysis with anti-HA. “Beads only” indicates a negative control whereby total extracts were incubated at 4°C in the absence of antibody.

Figure 3.

The C Terminus of RIN4 Is Required for NDR1 Interaction. Coimmunoprecipitation of HA:NDR1 and the N-terminal half (i.e., T7:RIN4-N) and C-terminal half (i.e., T7:RIN4-C) of RIN4. Immunoblot of anti-HA and anti-T7 immunoprecipitated proteins isolated 40 h after inoculation from wild-type N. benthamiana leaves hand-infiltrated with Agrobacterium strains expressing HA:NDR1 and the N- and C-terminal halves (separately) of RIN4. Total protein extracts were immunoprecipitated with anti-HA (NDR1) and anti-T7 (RIN4) antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti-HA (NDR1) and anti-T7 (RIN4) antibodies. The top two panels represent the absence of HA:NDR1-T7:RIN-N coimmunoprecipitation. The bottom two panels represent the HA:NDR1-T7:RIN4-C coimmunoprecipitation.

Figure 4.

The C-Terminal RIN4 Cleavage Products Differentially Associate with RPS2 and NDR1. Coimmunoprecipitation of RPS2:HA and HA:NDR1 with T7:RIN4_CLV-1, T7:RIN4_CLV-2, and T7:RIN4_CLV-3. Immunoblot of anti-HA and anti-T7 immunoprecipitated proteins isolated 24 h and 40 h after inoculation from wild-type N. benthamiana leaves hand-infiltrated with Agrobacterium strains expressing RPS2:HA, HA:NDR1, and RIN4-like cleavage products. Total protein extracts were immunoprecipitated with anti-HA (RPS2 and NDR1) and anti-T7 (RIN4) antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti-T7 (left panels) and anti-HA (right panels) antibodies. (A) RPS2:HA + T7:RIN4 cleavage products. (B) HA:NDR1 + T7:RIN4 cleavage products.

Figure 5.

The NDR1–RIN4 Interaction Occurs within the First Four Amino Acids of NDR1. Stepwise deletions of two and four amino acids from the N terminus of NDR1 results in a differential pattern of association with RIN4. (A) Coimmunoprecipitation and protein gel blot analysis of T7:NDR1 and HA:RIN4 from total protein extracts isolated from transiently expressed N. benthamiana. Loss of the NDR1–RIN4 interaction occurs following deletion of four amino acids from the N terminus of NDR1. (B) Homologous expression of the NDR1Δ4 deletion mutant in Arabidopsis. Native promoter expression of T7:NDR1 and T7:NDR1Δ4 cDNAs in the Arabidopsis ndr1-1 mutant background confirms results of transient heterologous expression. Native RIN4 antibodies coimmunoprecipitate T7:NDR1 yet fail to coimmunoprecipitate T7:NDR1Δ4. (C) Ultracentrifugation and localization of NDR1 and NDR1 deletion constructs confirm membrane localization. Total protein extracts from Arabidopsis plants (ndr1-1/HA:NDR1) expressing wild-type and deletion constructs were subjected to ultracentrifugation and separation by SDS-PAGE. Protein gel blot analysis (anti-T7) confirms plasma membrane localization for wild-type, Δ2, and Δ4 T7:NDR1 constructs.

Figure 6.

Gln and Asn at Positions 4 and 5 in NDR1 Are Required for RIN4 Association. Ala scanning of the first eight amino acids of NDR1 by QuickChange PCR mutagenesis. Sequential, independent changes in the native amino acid residues within the N-terminal tail of NDR1 demonstrate that the Gln and Asn residues at positions 4 and 5, respectively, are required for association with RIN4, as determined by coimmunoprecipitation experiments. NDR1 mutant constructs were transiently coexpressed with wild-type RIN4 in N. benthamiana for 40 h and processed as described previously. The sequence changes, where applicable, are indicated to the left of the protein gel blots as italicized and underlined letters. Anti-T7 blots (NDR1) are shown in the left column. Reciprocal, anti-HA blots (RIN4) are shown at the right.

Figure 7.

Construction of a Homologous Expression System for Characterizing the RPS2–RIN4–NDR1 Interaction. (A) rps2/ndr1-1 mutant Arabidopsis plants were transformed with a dual promoter construct expressing RPS2:HA and T7:NDR1/T7:NDR1Δ4 expressed under the control of their respective native promoters. (B) Protein gel blot analysis of RPS2:HA/T7:NDR1 and RPS2:HA/T7:NDR1Δ4 transgenic plants. Total protein extracts were separated by SDS-PAGE and probed with anti-HA antibodies (RPS2) and anti-T7 (NDR1) to determine protein expression levels in complemented Arabidopsis lines. (C) High-density (10⁵ colony-forming units [cfu]/mL) inoculation of wild-type Col-0 (top panels), RPS2:HA/T7:NDR1 (middle panels), and RPS2:HA/T7:NDR1 (bottom panels) with P. syringae DC3000 expressing empty vector (EV; control), AvrRpt2, AvrB, and AvrPphB effector proteins. (D) Bacterial growth curve analysis of native promoter expression lines of Arabidopsis RPS2:HA/T7:NDR1- and RPS2:HA/T7:NDR1Δ4-complemented plants. Bacterial counts were determined at days 0 and 4 following inoculation with P. syringae strains (10⁴ cfu/mL) expressing the indicated effector protein. Day 0 controls were plated 1 h after inoculation and represent the average bacterial growth count of all plant genotypes tested. The Arabidopsis ndr1-1/rps2 genotype corresponds to the parent genotype of the complemented expression system shown in (A). Data shown are the mean of four independent lines for both RPS2:HA/T7:NDR1 and RPS2:HA/T7:NDR1Δ4 constructs. Error bars indicate standard deviation calculated from the mean of replicate samples.