Catalytic domain of the diversified Pseudomonas syringae type III effector HopZ1 determines the allelic specificity in plant hosts

The type III secretion systems (T3SS) and secreted effectors (T3SEs) are essential virulence factors in Gram‐negative bacteria. During the arms race, plants have evolved resistance (R) genes to detect specific T3SEs and activate defence responses. However, this immunity can be efficiently defeated by the pathogens through effector evolution. HopZ1 of the plant pathogen Pseudomonas syringae is a member of the widely distributed YopJ T3SE family. Three alleles are known to be present in P. syringae, with HopZ1a most resembling the ancestral allelic form. In this study, molecular mechanisms underlying the sequence diversification‐enabled HopZ1 allelic specificity is investigated. Using domain shuffling experiments, we present evidence showing that a central domain upstream of the conserved catalytic cysteine residue determines HopZ1 recognition specificity. Random and targeted mutagenesis identified three amino acids involved in HopZ1 allelic specificity. Particularly, the exchange of cysteine141 in HopZ1a with lysine137 at the corresponding position in HopZ1b abolished HopZ1a recognition in soybean. This position is under strong positive selection, suggesting that the cysteine/lysine mutation might be a key step driving the evolution of HopZ1. Our data support a model in which sequence diversification imposed by the plant R gene‐associated immunity has driven HopZ1 evolution by allowing allele‐specific substrate‐binding.     

The evolution of Pseudomonas syringae host specificity and type III effector repertoires

The discovery 45 years ago that many Pseudomonas syringae pathovars elicit the hypersensitive response in plant species other than their hosts fostered the use of these bacteria as experimental models. However, the basis for host specificity and the corresponding resistance of nonhosts remain unclear. Pseudomonas syringae is now known to inject into the host cytoplasm, via the type III secretion system, effector proteins that suppress basal innate immunity, but may be recognized by cognate resistance (R) proteins in a second level of defence. The identification and manipulation of complete repertoires of type III effectors have revealed the highly polymorphic nature of effector repertoires and their potential to limit the host range. However, the maintenance of compatible effector repertoires may be driven by adaptations to life in a given plant species involving many factors. Tools are now available to test several hypotheses for the nature and evolution of P. syringae host specificity and nonhost resistance.     

The Pseudomonas syringae type III effector HopG1 targets mitochondria,alters plant development and suppresses plant innate immunity

The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector‐triggered immunity (ETI) and pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N‐terminal two‐thirds was sufficient for mitochondrial localization. A HopG1–GFP fusion lacking HopG1's N‐terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N‐terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1's target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions.     

The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.     

Pseudomonas syringae type III effector repertoires: last words in endless arguments

Many plant pathogens subvert host immunity by injecting compositionally diverse but functionally similar repertoires of cytoplasmic effector proteins. The bacterial pathogen Pseudomonas syringae is a model for exploring the functional structure of such repertoires. The pangenome of P. syringae encodes 57 families of effectors injected by the type III secretion system. Distribution of effector genes among phylogenetically diverse strains reveals a small set of core effectors targeting antimicrobial vesicle trafficking and a much larger set of variable effectors targeting kinase-based recognition processes. Complete disassembly of the 28-effector repertoire of a model strain and reassembly of a minimal functional repertoire reveals the importance of simultaneously attacking both processes. These observations, coupled with growing knowledge of effector targets in plants, support a model for coevolving molecular dialogs between effector repertoires and plant immune systems that emphasizes mutually-driven expansion of the components governing recognition.     

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.     

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.     

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.     

The Pseudomonas syringae type III effector tyrosine phosphatase HopAO1 suppresses innate immunity in Arabidopsis thaliana

The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects tomato and Arabidopsis plants, and is a model for studying the molecular basis of bacterial disease. Pst DC3000 secretes a battery of largely uncharacterized effector proteins into host cells via a type-III secretion system (TTSS). Little is currently known about the molecular mechanisms by which individual TTSS effectors promote virulence. The effector HopAO1 has similarity to protein tyrosine phosphatases, including a conserved catalytic site, and suppresses the hypersensitive response (HR) in some non-host plants. Whether HopAO1 has a similar effect in the host Arabidopsis is not clear. Here, we show that transgenic expression of HopAO1 in Arabidopsis suppresses callose deposition elicited by the Pst DC3000 hrpA mutant, and allows the normally non-pathogenic hrpA mutant to multiply within the leaf tissue. HopAO1 also suppresses resistance to Pst DC3000 induced by flg22, a pathogen-associated molecular pattern (PAMP). However, HopAO1 does not suppress the HR triggered by several classical avirulence genes. These results suggest that HopAO1 targets primarily PAMP-induced innate immunity in Arabidopsis. The virulence function of HopAO1 is dependent on an intact phosphatase catalytic site, as transgenic plants expressing a catalytically inactive derivative do not show these effects. Intriguingly, expression of the catalytically inactive HopAO1 has a dominant-negative effect on the function of the wild-type HopAO1. Analysis of mitogen-activated protein kinase (MAPK) activity suggests that HopAO1 targets a step downstream or independent of MAPK activation. Genome-wide expression analysis revealed that expression of several well-known defense genes was suppressed in hrpA mutant-infected HopAO1 transgenic plants.     

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses

The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen‐induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ‐glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.     

Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a.     

Structural analysis of Pseudomonas syringae AvrPtoB bound to host BAK1 reveals two similar kinase-interacting domains in a type III Effector

To infect plants, Pseudomonas syringae pv. tomato delivers ~30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution.     

RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis

In Arabidopsis, RPM1 confers resistance against Pseudomonas syringae expressing either of two sequence unrelated type III effectors, AvrRpm1 or AvrB. An RPM1-interacting protein (RIN4) coimmunoprecipitates from plant cell extracts with AvrB, AvrRpm1, or RPM1. Reduction of RIN4 protein levels inhibits both the hypersensitive response and the restriction of pathogen growth controlled by RPM1. RIN4 reduction causes diminution of RPM1. RIN4 reduction results in heightened resistance to virulent Peronospora parasitica and P. syringae, and ectopic defense gene expression. Thus, RIN4 positively regulates RPM1-mediated resistance yet is, formally, a negative regulator of basal defense responses. AvrRpm1 and AvrB induce RIN4 phosphorylation. This may enhance RIN4 activity as a negative regulator of plant defense, facilitating pathogen growth. RPM1 may "guard" against pathogens that use AvrRpm1 and AvrB to manipulate RIN4 activity.     

Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease

Upon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein. A structure-prediction method was employed in order to investigate possible biochemical functions of AvrRpt2. Results of a secondary structure prediction algorithm suggest that the functional C-terminal portion of AvrRpt2 is a cysteine protease. Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2-specific resistance response. These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.     

Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast

The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.     

Multiple approaches to a complete inventory of Pseudomonas syringae pv. tomato DC3000 type III secretion system effector proteins

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that translocates virulence effector proteins into host cells via a type III secretion system (T3SS). Many effector-encoding hypersensitive response and pathogenicity (Hrp) outer protein (hop) genes have been identified previously in DC3000 using bioinformatic methods based on Hrp promoter sequences and characteristic N-terminal amino acid patterns that are associated with T3SS substrates. To approach completion of the Hop/effector inventory in DC3000, 44 additional candidates were tested by the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter assay; 10 of the high-probability candidates were confirmed as T3SS substrates. Several previously predicted hop genes were tested for their ability to be expressed in an HrpL-dependent manner in culture or to be expressed in planta. The data indicate that DC3000 harbors 53 hop/avr genes and pseudogenes (encoding both injected effectors and T3SS substrates that probably are released to the apoplast); 33 of these genes are likely functional in DC3000, 12 are nonfunctional members of valid Hop families, and 8 are less certain regarding their production at functional levels. Growth of DC3000 in tomato and Arabidopsis Col-0 was not impaired by constitutive expression of repaired versions of two hops that were disrupted naturally by transposable elements or of hop genes that are naturally cryptic. In summary, DC3000 carries a complex mixture of active and inactive hop genes, and the hop genes in P. syringae can be identified efficiently by bioinformatic methods; however, a precise inventory of the subset of Hops that are important in pathogenesis awaits more knowledge based on mutant phenotypes and functions within plants.     

The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 encodes a type III chaperone and a cognate effector

Type III secretion systems are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcF(Pto) and HopF(Pto), that are preceded by a single hrp box promoter. We present evidence here to show that ShcF(Pto) and HopF(Pto) encode a type III chaperone and a cognate effector, respectively. ShcF(Pto) interacts with and stabilizes the HopF(Pto) protein in the bacterial cell. Translation of HopF(Pto) starts at a rare initiation codon ATA that limits the synthesis of the HopF(Pto) protein to a low level in bacterial cells.     

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.