David Mackey

Arabidopsis RIN4 Is a Target of the Type III Virulence Effector AvrRpt2 and Modulates RPS2-Mediated Resistance

Type III pili deliver effector proteins (virulence factors) from bacterial pathogens to host cells. Plants express disease resistance (R) proteins that respond specifically to a particular type III effector by activating immune responses. We demonstrated previously that two unrelated type III effectors from Pseudomonas syringae target and modify the Arabidopsis RIN4 protein. Here, we show that AvrRpt2, a third, unrelated type III effector, also targets RIN4 and induces its posttranscriptional disappearance. This effect is independent of the presence of RPS2, the Arabidopsis R protein that senses AvrRpt2. RIN4 overexpression inhibits multiple phenotypes associated with AvrRpt2 function. Conversely, disruption of RIN4 results in RPS2-dependent lethality. RPS2 and RIN4 physically associate in the plant. We suggest that RIN4 is the target of the AvrRpt2 virulence function, and that perturbation of RIN4 activates RPS2. Thus, RIN4 is a point of convergence for the activity of at least three unrelated P. syringae type III effectors.

Two Pseudomonas syringae Type III Effectors Inhibit RIN4-Regulated Basal Defense in Arabidopsis

Plant cells have two defense systems that detect bacterial pathogens. One is a basal defense system that recognizes complex pathogen-associated molecular patterns (PAMPs). A second system uses disease-resistance (R) proteins to recognize type lll effector proteins that are delivered into the plant cell by the pathogens type III secretion system. Here we show that these two pathways are linked. We find that two Pseudomonas syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the hosts basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1 for degradation and phosphorylation, respectively. We find that RIN4 is itself a regulator of PAMP signaling. The R proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems.

Arabidopsis RIN4 Negatively Regulates Disease Resistance Mediated by RPS2 and RPM1 Downstream or Independent of the NDR1 Signal Modulator and Is Not Required for the Virulence Functions of Bacterial Type III Effectors AvrRpt2 or AvrRpm1

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effectors host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.

MAMPs and MIMPs: proposed classifications for inducers of innate immunity

Plants encode a sophisticated innate immune system. Resistance against potential pathogens often relies on active responses. Prerequisite to the induction of defences is recognition of the pathogenic threat. Significant advances have been made in our understanding of the non‐self molecules that are recognized by plants and the means by which plants perceive them. Established terms describing these recognition events, including microbe‐associated molecular pattern (MAMP), MAMP‐receptor, effector, and resistance (R) protein, need clarification to represent our current knowledge adequately. In this review we propose criteria to classify inducers of plant defence as either MAMPs or microbe‐induced molecular patterns (MIMPs). We refine the definition of MAMP to mean a molecular sequence or structure in ANY pathogen‐derived molecule that is perceived via direct interaction with a host defence receptor. MIMPs are modifications of host‐derived molecules that are induced by an intrinsic activity of a pathogen‐derived effector and are perceived by a host defence receptor. MAMP‐receptors have previously been classified separately from R‐proteins as a discrete class of surveillance molecules. However, MAMP‐receptors and R‐proteins cannot be distinguished on the basis of their protein structures or their induced responses. We propose that MAMP‐receptors and MIMP‐receptors are each a subset of R‐proteins. Although our review is based on examples from plant pathogens and plants, the principles discussed might prove applicable to other organisms.

Specific Threonine Phosphorylation of a Host Target by Two Unrelated Type III Effectors Activates a Host Innate Immune Receptor in Plants

The Arabidopsis NB-LRR immune receptor RPM1 recognizes the Pseudomonas syringae type III effectors AvrB or AvrRpm1 to mount an immune response. Although neither effector is itself a kinase, AvrRpm1 and AvrB are known to target Arabidopsis RIN4, a negative regulator of basal plant defense, for phosphorylation. We show that RIN4 phosphorylation activates RPM1. RIN4(142-176) is necessary and, with appropriate localization sequences, sufficient to support effector-triggered RPM1 activation, with the threonine residue at position 166 being critical. Phosphomimic substitutions at T166 cause effector-independent RPM1 activation. RIN4 T166 is phosphorylated in vivo in the presence of AvrB or AvrRpm1. RIN4 mutants that lose interaction with AvrB cannot be coimmunoprecipitated with RPM1. This defines a common interaction platform required for RPM1 activation by phosphorylated RIN4 in response to pathogenic effectors. Conservation of an analogous threonine across all RIN4-like proteins suggests a key function for this residue beyond the regulation of RPM1.

The Coronatine Toxin of Pseudomonas syringae Is a Multifunctional Suppressor of Arabidopsis Defense

The bacterial phytotoxin coronatine mimics a plant hormone, jasmonic acid, and thus antagonizes signaling by another plant hormone, salicylic acid, important for plant defense. This study demonstrates that coronatine also suppresses SA-independent host defense, including the production of defense-promoting indole glucosinolates and has a target other than the jasmonic acid receptor. The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box–containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.

Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense

Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone‐responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2‐DE facilitated the discovery of low abundance proteins – enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA‐binding protein, and a C2‐domain‐containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA‐binding protein, involvement of PTM and post‐transcriptional regulation are implicated, respectively.

WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability.

The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system.

The phytotoxin coronatine is a multifunctional component of the virulence armament of Pseudomonas syringae

Plant pathogens deploy an array of virulence factors to suppress host defense and promote pathogenicity. Numerous strains of Pseudomonas syringae produce the phytotoxin coronatine (COR). A major aspect of COR function is its ability to mimic a bioactive jasmonic acid (JA) conjugate and thus target the JA-receptor COR-insensitive 1 (COI1). Biological activities of COR include stimulation of JA-signaling and consequent suppression of SA-dependent defense through antagonistic crosstalk, antagonism of stomatal closure to allow bacterial entry into the interior of plant leaves, contribution to chlorotic symptoms in infected plants, and suppression of plant cell wall defense through perturbation of secondary metabolism. Here, we review the virulence function of COR, including updates on these established activities as well as more recent findings revealing COI1-independent activity of COR and shedding light on cooperative or redundant defense suppression between COR and type III effector proteins.

Contrasting Roles of the Apoplastic Aspartyl Protease APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis Systemic Acquired Resistance,

Proteins accumulating in the apoplast of Arabidopsis during the emission of systemic immune signals include an aspartyl protease that dampens systemic acquired resistance and a legume lectin-like protein that promotes it. Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance.