Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait

We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.     

The HopZ family of Pseudomonas syringae type III effectors require myristoylation for virulence and avirulence functions in Arabidopsis thaliana

Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.     

Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae

Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems. Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts. Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts. To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P. syringae type III effectors and two effector chaperones. We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer. We assessed the role of diversifying and purifying selection in the evolution of these gene families. While some P. syringae type III effectors were acquired recently, others have evolved predominantly by descent. The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function. However, members of 7 families had domains subject to diversifying selection.     

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.     

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 effector's 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.     

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 pathogen's 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 host's 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.     

Layered basal defenses underlie non-host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola

Arabidopsis is a non-host for Pseudomonas syringae pv. phaseolicola NPS3121 (Pph), a bacterial pathogen of bean. Pph does not induce a hypersensitive response in Arabidopsis. Here we show that Arabidopsis instead resists Pph with multi-layered basal defense. Our approach was: (i) to identify defense readouts induced by Pph; (ii) to determine whether mutations in known Arabidopsis defense genes disrupt Pph-induced defense signaling; (iii) to determine whether heterologous type III effectors from pathogens of Arabidopsis suppress Pph-induced defense signaling, and (iv) to ascertain how basal defenses contribute to resistance against Pph by individually or multiply disrupting defense signaling pathways with mutations and heterologous type III effectors. We demonstrate that Pph elicits a minimum of three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 protein accumulation and morphologically distinct types of callose deposition. Further, they require distinct defense genes, including PMR4, RAR1, SID2, NPR1, and PAD4 . Finally, they are suppressed differentially by heterologous type III effectors, including AvrRpm1 and HopM1. Pph growth is enhanced only when multiple defense pathways are disrupted. For example, mutation of NPR1 or SID2 combined with the action of AvrRpm1 and HopM1 renders Arabidopsis highly susceptible to Pph. Thus, non-host resistance of Arabidopsis to Pph is based on multiple, individually effective layers of basal defense.     

The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.     

The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into plant cells via a type III secretion system (T3SS), which is required for pathogenesis. The protein HrpJ is secreted by P. syringae and is required for a fully functional T3SS. A hrpJ mutant is non-pathogenic and cannot inject effectors into plant cells or secrete the harpin HrpZ1. Here we show that the hrpJ mutant also cannot secrete the harpins HrpW1 and HopAK1 or the translocator HrpK1, suggesting that these proteins are required in the translocation (injection) of effectors into plant cells. Complementation of the hrpJ mutant with secretion incompetent HrpJ derivatives restores the secretion of HrpZ1 and HrpW1 and the ability to elicit a hypersensitive response, a measure of translocation. However, growth in planta and disease symptom production is only partially restored, suggesting that secreted HrpJ may have a direct role in virulence. Transgenic Arabidopsis plants expressing HrpJ-HA complemented the virulence phenotype of the hrpJ mutant expressing a secretion incompetent HrpJ derivative and were reduced in their immune responses. Collectively, these data indicate that HrpJ has a dual role in P. syringae: inside bacterial cells HrpJ controls the secretion of translocator proteins and inside plant cells it suppresses plant immunity.     

Antagonism between salicylic and abscisic acid reflects early host–pathogen conflict and moulds plant defence responses

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant–pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana . We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.     

The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non-host plants

The bacterial plant pathogen Pseudomonas syringae injects a large repertoire of effector proteins into plant cells using a type III secretion apparatus. Effectors can trigger or suppress defences in a host-dependent fashion. Host defences are often accompanied by programmed cell death, while interference with defences is sometimes associated with cell death suppression. We previously predicted the effector repertoire of the sequenced bean pathogen P. syringae pv. syringae ( Psy ) B728a using bioinformatics. Here we show that Psy B728a is also pathogenic on the model plant species Nicotiana benthamiana (tobacco). We confirm our effector predictions and clone the nearly complete Psy B728a effector repertoire. We find effectors to have different cell death-modulating activities and distinct roles during the infection of the susceptible bean and tobacco hosts. Unexpectedly, we do not find a strict correlation between cell death-eliciting and defence-eliciting activity and between cell death-suppressing activity and defence-interfering activity. Furthermore, we find several effectors with quantitative avirulence activities on their susceptible hosts, but with growth-promoting effects on Arabidopsis thaliana , a species on which Psy B728a does not cause disease. We conclude that P. syringae strains may have evolved large effector repertoires to extend their host ranges or increase their survival on various unrelated plant species.     

A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae

The injection of nearly 30 effector proteins by the type III secretion system underlies the ability of Pseudomonas syringae pv. tomato DC3000 to cause disease in tomato and other host plants. The search for effector functions is complicated by redundancy within the repertoire and by plant resistance (R)-gene sentinels, which may convert effector virulence activities into a monolithic defense response. On the premise that some effectors target universal eukaryotic processes and that yeast (Saccharomyces cerevisiae) lacks R genes, the DC3000 effector repertoire was expressed in yeast. Of 27 effectors tested, HopAD1, HopAO1, HopD1, HopN1, and HopU1 were found to inhibit growth when expressed from a galactose-inducible GAL1 promoter, and HopAA1-1 and HopAM1 were found to cause cell death. Catalytic site mutations affecting the tyrosine phosphatase activity of HopAO1 and the cysteine protease activity of HopN1 prevented these effectors from inhibiting yeast growth. Expression of HopAA1-1, HopAM1, HopAD1, and HopAO1 impaired respiration in yeast, as indicated by tests with ethanol glycerol selective media. HopAA1-1 colocalized with porin to yeast mitochondria and was shown to cause cell death in yeast and plants in a domain-dependent manner. These results support the use of yeast for the study of plant-pathogen effector repertoires.     

The Pseudomonas syringae type III effector AvrRpt2 functions downstream or independently of SA to promote virulence on Arabidopsis thaliana

AvrRpt2, a Pseudomonas syringae type III effector protein, functions from inside plant cells to promote the virulence of P. syringae pv. tomato strain DC3000 (PstDC3000) on Arabidopsis thaliana plants lacking a functional copy of the corresponding RPS2 resistance gene. In this study, we extended our understanding of AvrRpt2 virulence activity by exploring the hypothesis that AvrRpt2 promotes PstDC3000 virulence by suppressing plant defenses. When delivered by PstDC3000, AvrRpt2 suppresses pathogen-related (PR) gene expression during infection, suggesting that AvrRpt2 suppresses defenses mediated by salicylic acid (SA). However, AvrRpt2 promotes PstDC3000 growth on transgenic plants expressing the SA-degrading enzyme NahG, indicating that AvrRpt2 does not promote bacterial virulence by modulating SA levels during infection. AvrRpt2 general virulence activity does not depend on the RPM1 resistance gene, as mutations in RPM1 had no effect on AvrRpt2-induced phenotypes. Transgenic plants expressing AvrRpt2 displayed enhanced susceptibility to PstDC3000 strains defective in type III secretion, indicating that enhanced susceptibility of these plants is not because of suppression of defense responses elicited by other type III effectors. Additionally, avrRpt2 transgenic plants did not exhibit increased susceptibility to Peronospora parasitica and Erysiphe cichoracearum, suggesting that AvrRpt2 virulence activity is specific to P. syringae.     

Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells

Many bacterial pathogens cause disease by injecting virulence proteins (effectors) into host cells via the specialized type III secretion system. Recently, exceptional progress in identifying effectors was made in the phytopathogen Pseudomonas syringae using a novel genetic screen and bioinformatic approach. These studies, along with localization experiments, suggest that most P. syringae effectors function by targeting the plasma membrane, chloroplasts or mitochondria of host cells. The type III secretome of P. syringae is highly variable and dynamic, a lesson gleaned from a comparative genomic analysis. Variation in the effector repertoire is likely to facilitate the adaptation of P. syringae to different hosts.     

AlgW regulates multiple Pseudomonas syringae virulence strategies

Gram-negative bacterial pathogens have evolved a number of virulence-promoting strategies including the production of extracellular polysaccharides such as alginate and the injection of effector proteins into host cells. The induction of these virulence mechanisms can be associated with concomitant downregulation of the abundance of proteins that trigger the host immune system, such as bacterial flagellin. In Pseudomonas syringae, we observed that bacterial motility and the abundance of flagellin were significantly reduced under conditions that induce the type III secretion system. To identify genes involved in this negative regulation, we conducted a forward genetic screen with P. syringae pv. maculicola ES4326 using motility as a screening phenotype. We identified the periplasmic protease AlgW as a key negative regulator of flagellin abundance that also positively regulates alginate biosynthesis and the type III secretion system. We also demonstrate that AlgW constitutes a major virulence determinant of P. syringae required to dampen plant immune responses. Our findings support the conclusion that P. syringae co-ordinately regulates virulence strategies through AlgW in order to effectively suppress host immunity.     

Type III protein secretion in Pseudomonas syringae

The type III secretion system is an essential virulence system used by many Gram-negative bacterial pathogens to deliver effector proteins into host cells. This review summarizes recent advancements in the understanding of the type III secretion system of Pseudomonas syringae, including regulation of the type III secretion genes, assembly of the Hrp pilus, secretion signals, the putative type III effectors identified to date, and their virulence action after translocation into plant cells.