Pathogen Effectors Target Arabidopsis EDS1 and Alter Its Interactions with Immune Regulators

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Science  09 Dec 2011:
Vol. 334, Issue 6061, pp. 1405-1408
DOI: 10.1126/science.1211592

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Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll–interleukin-1 receptor–nucleotide binding–leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR–mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.

Like other organisms, plants tightly control the onset and amplitude of potent immune responses to pathogens for optimal growth and development (1, 2). The first step in inducible defenses, perception of a pathogen and elicitation of basal resistance, is suppressed by successful pathogens that deploy effectors in the host cell (3). As a second line of innate immunity, plants sense injected effectors through resistance proteins that generally are specific to individual effectors (47). In the absence of an adaptive immune system, this innate effector-triggered immune response is key for plants to defeat coevolved adapted pathogens. Both direct and indirect detection of effectors by resistance proteins have been documented (6). In the latter case, resistance proteins associate with host proteins that are effector virulence targets or their decoys. Detecting biochemical activity of effectors, rather than effector epitopes, reduces the number of resistance proteins a plant has to deploy to protect against a potentially vast number of structurally diverse and rapidly evolving effectors (5, 7).

Early molecular events that signal effector-triggered immunity activation consist of changes in intra- and intermolecular interactions of resistance proteins (1, 8). Genetic approaches have identified only a few genes considered to encode signaling proteins downstream of resistance proteins. Among these, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is a central regulator of basal resistance, and of effector-triggered immunity mediated by resistance proteins belonging to the Toll–interleukin-1 receptor–nucleotide binding–leucine-rich repeat (TIR-NB-LRR) class of resistance proteins (911). In contrast, coiled-coil (CC)-NB-LRR proteins do not require EDS1 (9). One possible reason for the paucity of identified effector-triggered immunity signaling proteins is a short signaling pathway. Several resistance proteins, including RESISTANCE TO PSEUDOMONAS SYRINGAE4 (RPS4), localize to the nucleus (12, 13), and the TIR-NB-LRR protein SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1) binds the transcriptional repressor TOPLESS-RELATED1 (TPR1) in the nucleus, leading to changes in defense gene expression (14). The identity and true extent of the plant effector-triggered immunity signaling pathway(s) are major open questions (6). Here we identify EDS1 as a target for the sequence-unrelated bacterial effectors AvrRps4 and HopA1, which genetically are detected by the EDS1-dependent resistance genes RPS4 and RPS6, respectively.

Our present study originated with SUPPRESSOR OF rps4-RLD1 (SRFR1), which genetically functions as a negative regulator of effector-triggered immunity and encodes a tetratricopeptide repeat domain protein widely conserved among eukaryotic organisms. Mutations in SRFR1 enhance resistance to bacteria expressing AvrRps4 or HopA1 when the corresponding resistance gene RPS4 or RPS6, respectively, is not functional (15, 16). This enhanced resistance is dependent on EDS1. In the accession Columbia-0 (Col-0) with the srfr1-4 knock-out allele, the Col-0–specific SNC1 gene is activated, leading to non-specific enhanced resistance to virulent Pseudomonas syringae pv. tomato strain DC3000 (DC3000) (17) and Hyaloperonospora arabidopsidis isolates (18).

Because not all enhanced resistance phenotypes are abolished in a srfr1-4 snc1-11 double mutant (17), we explored whether additional TIR-NB-LRR genes are activated in srfr1-4 by crossing it to eds1-2 (19). F2 progeny homozygous for srfr1-4 and with at least one wild-type copy of EDS1 were stunted, whereas srfr1-4 eds1-2 double-homozygous plants were indistinguishable from wild-type Col-0 (Fig. 1A). In addition, srfr1-4 eds1-2 plants displayed complete loss of increased basal resistance to DC3000 and of constitutive up-regulation of defense genes (Fig. 1, B and C), which included the up-regulation of resistance genes in srfr1-4 (fig. S1) that may be the basis for constitutively activated defenses (17). The srfr1-4 eds1-2 plants also lost resistance to DC3000 expressing AvrRps4 or HopA1, but not AvrRpm1 (fig. S2), which is consistent with published data (9, 16). Collectively, these results show the EDS1 dependence of heightened resistance in srfr1-4 plants and indicate that additional TIR-NB-LRR genes in srfr1-4 snc1-11 contribute to constitutive activation of defenses.

Fig. 1

Constitutive defense phenotypes of srfr1-4 depend on a functional EDS1 allele. (A) Stunted growth of srfr1-4 is abolished in srfr1-4 eds1-2 plants. Four-week-old plants are shown. (B) Enhanced basal resistance of srfr1-4 plants to DC3000 is abolished in srfr1-4 eds1-2 plants. Graph represents in planta bacterial growth of DC3000 inoculated at a bacterial density of 5 × 104 colony-forming units (CFU)/ml at 24°C. (C) eds1-2 abolishes increased defense gene expression in srfr1-4 plants. SID2, EDS1, SRFR1, PAD4 (left), and PR1 (right) mRNA levels were normalized to SAND gene (At2g28390) expression and represent averages of three biological replicates. In (B) and (C), error bars denote standard deviation. Different letters above the bars indicate statistically significant differences determined by Student’s t test (P < 0.05). Assay in (B) was repeated once and in (C) twice with similar results.

Based on this finding and the multiple intersections of SRFR1 and EDS1 regulatory pathways in TIR-NB-LRR–mediated effector-triggered immunity, we investigated molecular interactions between EDS1, SRFR1, and the TIR-NB-LRR resistance proteins RPS4, SNC1, and RPS6. We first used bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana (19), which indeed detected EDS1 interactions with SRFR1. EDS1-SRFR1 complexes mainly localized to punctate spots in the cytoplasm (Fig. 2A and fig. S3A), similar to localization of SRFR1 (16). EDS1 complexes with RPS4, SNC1, and RPS6 showed localization patterns similar to that of EDS1-SRFR1 that were distinct from the diffuse EDS1-PAD4 (PHYTOALEXIN DEFICIENT4) localization (Fig. 2A and fig. S3A). No interactions were detected for EDS1 with the CC-NB-LRR resistance protein RPM1 (Fig. 2A and fig. S3A) or with β-glucuronidase (GUS) (fig. S3B). None of the resistance proteins or SRFR1 interacted with PAD4 (fig. S3B). We also detected EDS1 interactions with SRFR1, RPS4, and SNC1 in the nucleus (fig. S4).

Fig. 2

EDS1 interacts with SRFR1 and the TIR-NB-LRR resistance proteins RPS4, RPS6, and SNC1. (A) BiFC analysis 2 days after infiltration of N. benthamiana leaves. Red fluorescence is indicative of RFP whereas yellow fluorescence shows reconstitution of YFP as an indicator of protein-protein interactions (19). Arrowheads indicate position of nuclei. Scale bars, 20 μm. (B) Co-IP of proteins transiently expressed in N. benthamiana leaves. Immunoprecipitates (IP) were analyzed by immunoblot (IB) with the indicated antibodies. Results from cytoplasmic microsomal fractions are shown here. Input extracts for immunoprecipitates are shown in the bottom two panels. Molecular mass values (in kD) are shown on the left. Asterisks mark the expected sizes of HA-tagged proteins. (C) Co-IPs on stable transgenic Arabidopsis lines expressing EDS1-YFP and HA-SRFR1. Cytoplasmic soluble and microsomal fractions were subjected to IP with antibodies against GFP or HA(αGFP and αHA). Microsomal inputs are threefold enriched compared with soluble fractions. Fraction enrichment is indicated by IB assays with antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (soluble) or antibody against vacuolar type H+–adenosine triphosphatase (V-ATPase) (microsomal). This experiment was repeated three times with similar results.

We validated the observed protein-protein interactions by coimmunoprecipitation (co-IP) assays of transiently expressed proteins with a different set of epitope tags in N. benthamiana (19). These assays confirmed the interactions of EDS1 with SRFR1, SNC1, RPS4, and RPS6 in the cytoplasmic microsomal fraction. No interaction was observed with RPM1 (Fig. 2B). Further, we assayed for SRFR1-EDS1 interactions in Arabidopsis by transforming plants expressing EDS1-YFP (yellow fluorescent protein) with a genomic clone of hemagglutinin (HA) epitope–tagged SRFR1 (HA-SRFR1g) (19). Expression of both transgenes is driven by the respective native promoter, and both complement a corresponding mutant line (17, 20). In reciprocal co-IPs, we detected SRFR1-EDS1 interactions in the cytoplasmic microsomal fraction of these plants (Fig. 2C). This result and previous localizations of RPS4 to endomembrane fractions (13) indicate that the BiFC and co-IP assays in N. benthamiana reflected native protein interactions.

We also detected the presence of SRFR1-RPS6 in addition to SRFR1-RPS4 complexes in the cytoplasmic microsomal fractions, whereas SRFR1 did not interact with RPM1 in either fraction (fig. S5). The interactions documented so far therefore include the negative regulator SRFR1, the positive regulator EDS1, and specifically the TIR-NB-LRR resistance proteins SNC1, RPS4, and RPS6. Of these proteins, only SRFR1 and RPS4 are not also cytoplasmic soluble proteins, indicating that SRFR1 may be the protein that localizes these complexes to a microsomal membrane.

Based on the genetic and physical connections between these immune regulators and effectors, we hypothesized that EDS1 might be a common virulence target that is guarded by TIR-NB-LRR proteins, and tested interactions of AvrRps4 and HopA1 with EDS1. We detected reciprocal BiFC interactions of AvrRps4 with EDS1 in the cytoplasm and nucleus, and HopA1 with EDS1 in the cytoplasm, but not with AvrRpm1 and EDS1 (Fig. 3A). These data were further supported by co-IP assays in N. benthamiana. Consistent with reconstituted YFP localizations in BiFC, AvrRps4-EDS1 complexes were detected in the microsomal fraction, whereas HopA1-EDS1 complexes and EDS1 dimers were found both in the soluble and microsomal fraction (Fig. 3B). No interactions were observed with AvrRpm1 even though it was expressed to high levels, showing the specificity of EDS1-effector interactions (Fig. 3B). To test whether these EDS1-effector interactions are direct, we performed in vitro pull-downs of EDS1 with AvrpRps4 or HopA1 expressed in Escherichia coli (19). As shown in fig. S6, reciprocal pull-downs showed that EDS1 directly interacted with these sequence-unrelated effectors.

Fig. 3

TIR-NB-LRR resistance protein–specific effectors interact with EDS1. (A) BiFC analysis as described in Fig. 2A. Proteins listed first were fused to nVenus, and those listed second to the cerulean version of the cyan fluorescent protein, cCFP. Scale bar, 20 μm. (B) Co-IP analysis of cytoplasmic soluble and microsomal extracts from N. benthamiana plants transiently expressing Myc-EDS1 with HA-tagged EDS1, AvrRps4, AvrRpm1, or HopA1. Lower four panels show the input fractions for the IP assays. Microsomal inputs are threefold enriched compared with soluble inputs. Fraction enrichment was determined with antibodies against GAPDH and V-ATPase. Molecular mass values (in kD) are shown to the left. Asterisks denote expected sizes of full-length HA-tagged proteins. The filled circle indicates the processed form of AvrRps4 (29). This experiment was repeated once with similar results.

Because EDS1 specifically interacted with two effectors that cause EDS1-dependent effector-triggered immunity, we next tested whether EDS1-resistance protein complexes are a target of these effectors. N. benthamiana leaves expressing Myc-EDS1 and HA-SRFR1, HA-RPS4 or HA-RPS6 were challenged with green fluorescent protein (GFP) or GFP-tagged AvrRps4, HopA1, or AvrRpm1, and microsomal fractions were analyzed by co-IP. We observed that SRFR1-EDS1 interactions were significantly reduced in the presence of AvrRps4 and HopA1, whereas AvrRpm1 did not cause alterations (Fig. 4). We did not detect any changes in the localization of SRFR1-EDS1 complexes in the presence of effectors, and neither SRFR1-RPS4 nor SRFR1-RPS6 complexes were perturbed by effectors (fig. S7). Both AvrRps4 and HopA1, but not AvrRpm1, also effectively reduced EDS1-RPS4 and EDS1-RPS6 interactions (Fig. 4), consistent with EDS1 being the effector target and cross-talk between EDS1-associated TIR-NB-LRR proteins (17). AvrRps4 and HopA1 trigger immunity in Arabidopsis srfr1 mutants even when the corresponding resistance protein is absent. This resistance is likely triggered by other TIR-NB-LRR proteins that detect the effector's interaction with EDS1. Our competitive co-IP assays would favor enhanced cross-talk because endogenous N. benthamiana SRFR1 was likely underexpressed compared with the overexpressed EDS1, resistance proteins and effectors.

Fig. 4

TIR-NB-LRR resistance protein-specific effectors disrupt EDS1 interactions. Myc-EDS1 was transiently expressed either in HA-SRFR1g transgenic (top) or wild-type N. benthamiana with HA-RPS4 (middle) or HA-RPS6 (bottom) in combination with either GFP or GFP-tagged AvrRps4, HopA1, or AvrRpm1. Shown are microsomal fractions immunoprecipitated with antibody against HA. Input fractions show equal amounts of protein for each set of IPs. This experiment was repeated twice with similar results.

Our results lead to a model that parsimoniously integrates genetic data on EDS1 function, its tight association with the TIR-NB-LRR class of resistance genes, and its dual role in regulating basal resistance and effector-triggered immunity. In this model, a certain number of TIR-NB-LRR resistance proteins guard EDS1. As a central regulator of basal resistance with PAD4 and SAG101 (21, 22), EDS1 constitutes a biologically relevant target for effectors that attempt to disable EDS1 immune function. Perturbation of EDS1 complexes by effectors would then trigger activation of TIR-NB-LRR resistance proteins that interact with EDS1, and relocalization of EDS1 and resistance proteins. Similar to RIN4 (23), disruption of EDS1 complexes may be the result of post-translational modifications to EDS1 induced by effectors.

It was shown recently that EDS1 alone functions in effector-triggered immunity, whereas EDS1-PAD4 interactions are required for basal resistance (24), consistent with only EDS1, and not PAD4, interacting with resistance proteins tested here. We also did not observe disruption of EDS1-PAD4 interactions by AvrRps4 (fig. S8). Together, these data suggest that EDS1's main function is to signal in basal resistance, while its function in effector-triggered immunity derives from being guarded by TIR-NB-LRR proteins. This model does not exclude a role for EDS1 in regulating effector-triggered immunity downstream of resistance protein activation, since effector-triggered immunity and basal resistance responses are intertwined (25). Given that EDS1, PAD4 and SAG101 are sequence-related lipase-like proteins, one could propose that EDS1 is a decoy (26) to trap effectors that target PAD4 and SAG101. However, the decoy model would not explain the severe enhanced disease susceptibility phenotype of eds1 mutants that gave this gene its name and has been well-documented with diverse plant pathogens, or the conservation of EDS1 genes in monocotyledonous plants that do not possess TIR-NB-LRR genes.

Multiple TIR-NB-LRR resistance proteins guarding EDS1 can also explain cross-talk in TIR-NB-LRR signaling. The guard model of resistance protein specificity and indirect effector recognition was first substantiated with RIN4, a host protein targeted by the three sequence-unrelated effectors AvrRpm1, AvrB and AvrRpt2, and guarded by the CC-NB-LRR proteins RPM1 and RPS2 (6). Interestingly, it was shown that AvrRpm1 in the absence of its canonical resistance protein RPM1 can activate RPS2 in RIN4 RPS2 rpm1 plants, presumably because RIN4 is a common target of these effectors (27).

A new element in the EDS1 guard complex is SRFR1, genetically a negative regulator of TIR-NB-LRR resistance protein-mediated immunity. SRFR1 is likely to localize EDS1-resistance protein interactions to a microsomal compartment. Either by interacting with the resistance protein co-chaperone SGT1 (18) or by directly affecting the stability of EDS1-resistance protein complexes shown here, SRFR1 could set a threshold at which resistance proteins are activated, thus regulating resistance protein specificity. In the absence of SRFR1, EDS1-resistance protein complexes would be more easily perturbed by any effector targeting EDS1, resulting in increased cross-talk between TIR-NB-LRR proteins.

We propose that disruption by AvrRps4 of EDS1-RPS4 interactions at a cytoplasmic membrane, where RPS4 is predominantly found at resting state (13), constitutes the very first step in RPS4 activation. None of these interactors are integral membrane proteins. Interestingly, Heidrich et al. (28) observe that activated RPS4 together with EDS1 is predominantly found in the soluble cytoplasmic fraction. In the nucleus, EDS1 was also found to interact with AvrRps4 and RPS4. Furthermore, Heidrich et al. (28) show that similar to RPS4 and EDS1 (13, 20), AvrRps4 needs to be nuclear-localized for full immunity activation. A function of effector-triggered immunity activation is therefore likely to include alteration of protein complexes to generate a mobile signal to the nucleus. Which of the many TIR-NB-LRR proteins directly interact with EDS1 and how effectors eventually activate immune responses in the nucleus remains to be established.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

Table S1

References (3035)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank J. Parker for providing seed of eds1-2 and EDS1-YFP plants, F. Gao and J. C. Nam for technical assistance, and the University of Missouri Molecular Cytology Core for assistance with confocal fluorescence microscopy. This work was supported by the NSF Integrative Organismal Systems Program (grants IOS-0715926 and IOS-1121114) (W.G.).
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