The HopF family of Pseudomonas syringae type III secreted effectors

Pseudomonas syringae is a bacterial phytopathogen that utilizes the type III secretion system to inject effector proteins into plant host cells. Pseudomonas syringae can infect a wide range of plant hosts, including agronomically important crops such as tomatoes and beans. The ability of P. syringae to infect such numerous hosts is caused, in part, by the diversity of effectors employed by this phytopathogen. Over 60 different effector families exist in P. syringae; one such family is HopF, which contains over 100 distinct alleles. Despite this diversity, research has focused on only two members of this family: HopF1 from P. syringae pathovar phaseolicola 1449B and HopF2 from P. syringae pathovar tomato DC3000. In this study, we review the research on HopF family members, including their host targets and molecular mechanisms of immunity suppression, and their enzymatic function. We also provide a phylogenetic analysis of this expanding effector family which provides a basis for a proposed nomenclature to guide future research. The extensive genetic diversity that exists within the HopF family presents a great opportunity to study how functional diversification on an effector family contributes to host specialization.     

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.     

The Pseudomonas syringae type III effector AvrRpm1 induces significant defenses by activating the Arabidopsis nucleotide-binding leucine-rich repeat protein RPS2

Plant disease resistance (R) proteins recognize potential pathogens expressing corresponding avirulence (Avr) proteins through 'gene-for-gene' interactions. RPM1 is an Arabidopsis R-protein that triggers a robust defense response upon recognizing the Pseudomonas syringae effector AvrRpm1. Avr-proteins of phytopathogenic bacteria include type III effector proteins that are often capable of enhancing virulence when not recognized by an R-protein. In rpm1 plants, AvrRpm1 suppresses basal defenses induced by microbe-associated molecular patterns. Here, we show that expression of AvrRpm1 in rpm1 plants induced PR-1, a classical defense marker, and symptoms including chlorosis and necrosis. PR-1 expression and symptoms were reduced in plants with mutations in defense signaling genes ( pad4 , sid2 , npr1 , rar1 , and ndr1 ) and were strongly reduced in rpm1 rps2 plants, indicating that AvrRpm1 elicits defense signaling through the Arabidopsis R-protein, RPS2. Bacteria expressing AvrRpm1 grew more on rpm1 rps2 than on rpm1 plants. Thus, independent of its classical 'gene-for-gene' activation of RPM1, AvrRpm1 also induces functionally relevant defenses that are dependent on RPS2. Finally, AvrRpm1 suppressed host defenses and promoted the growth of type III secretion mutant bacteria equally well in rps2 and RPS2 plants, indicating that virulence activity of over-expressed AvrRpm1 predominates over defenses induced by weak activation of RPS2.     

Distinct Pseudomonas type‐III effectors use a cleavable transit peptide to target chloroplasts

The pathogen Pseudomonas syringae requires a type‐III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type‐III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type‐III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector‐triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP‐triggered immune responses compared with wild‐type plants. An N‐terminal region of HopK1 shares similarity with the corresponding region in the well‐studied type‐III effector AvrRps4; however, their C‐terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N‐terminal regions of these type‐III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4‐induced HR did not. Our results suggest that a primary virulence target of these type‐III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type‐III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire‐resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram‐negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease‐resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E‐deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C‐terminal truncated HopAZ1 abolished HopAZ1‐dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.     

Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)₅ or (SP)₃₂, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)_n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)₅ ≪ (SP)₅-EGFP < (SP)₃₂-EGFP. The EGFP equivalent yield of (SP)₃₂-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)₃₂-EGFP (∼115 kDa) and partially glycosylated (SP)₃₂-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.     

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram‐negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ^Xoo) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ^Xoo does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca²⁺ coordination shell of a ribose‐binding site and the spacious active site in XopQ^Xoo, we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ^Xoo. Here, we report the crystal structure of XopQ^Xoo in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca²⁺ concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ^Xoo with its ribosyl end tethered to the Ca²⁺ coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand‐induced conformational changes. These data showed that XopQ^Xoo is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ^Xoo. Proteins 2014; 82:2910–2914. © 2014 Wiley Periodicals, Inc.     

Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS⁺ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR⁺. Class II strains are T3SS^– or, if T3SS⁺, also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR⁺, produce robust chlorotic spots. Class III strains are T3SS⁺ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low‐level inoculum and confocal microscopy of fluorescent protein‐labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi‐quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.     

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors.     

丁香假单胞大豆致病变种两个III型效应因子的克隆及功能分析

为了研究Ⅲ型泌出效应因子在丁香假单胞大豆致病变种中的作用,利用反向PCR技术,首次从丁香假单胞大豆致病变种全基因组中克隆得到两个效应因子HopAB1和HopAF1基因的同源物,分别命名为HopAB1s和HopAF1s。生物信息学分析表明,HopAB1s基因全长是1 572 bp,编码523个氨基酸;HopAF1s基因全长是855 bp,编码284个氨基酸。即基因的登录号分别为JF826562和JF826563。保守功能区预测显示HopAB1s在N末端包含一个E3泛素连接酶功能区。将这2个基因克隆到PVX二元表达载体并转化农杆菌,利用农杆菌介导的瞬时侵染技术在本生烟中表达,发现2个效应因子均能抑制由鼠凋亡因子激发的细胞程序性死亡;将烟草疫霉接种在表达效应基因的区域,发现效应因子能促进烟草疫霉侵染烟草,因此本研究得到的两个效应因子是免疫抑制因子,为进一步研究该菌的致病机理奠定基础。     

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.     

Early events in the pathogenicity of Pseudomonas syringae on Nicotiana benthamiana

Conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns) elicit defence responses in plants through extracellular receptor proteins. One important PAMP is the flagellin protein derived from motile bacteria. We show here that the solanaceous species Nicotiana benthamiana perceives the flagellin proteins of both pathogenic and non-host species of Pseudomonas syringae. The response to flagellin required a gene closely related to that encoding the Arabidopsis thaliana flagellin receptor that we designated NbFls2. In addition, silencing of NbFls2 led to increased growth of compatible, non-host and non-pathogenic strains of P. syringae. Thus, flagellin perception restricts growth of P. syringae strains on N. benthamiana. Pathogenic bacteria secrete effector proteins into the plant cell to enhance virulence. We tested the ability of several unrelated effectors to suppress PAMP-mediated defences. The effector proteins AvrPto and AvrPtoB, but not AvrRps4, suppressed all responses tested including the hypersensitive response induced by non-host flagellins and the oomycete elicitor INF1. Strikingly, transient expression of avrPto or avrPtoB stimulated the growth of non-pathogenic Agrobacterium tumefaciensin planta, suggesting that multiplication of this species is also restricted by PAMP perception. Unexpectedly, AvrPtoB but not AvrPto required the defence-associated genes Rar1, Sgt1 and Eds1 for suppression. This observation separates the respective mechanisms of the two effectors, and suggests that AvrPtoB may target the defence machinery directly for its suppressive effect.     

Diversifying selection drives the evolution of the type III secretion system pilus of Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host's pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima's D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.     

A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.     

Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system

In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.     

Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs

The transient expression of recombinant biopharmaceutical proteins in plants can suffer inter‐batch variation, which is considered a major drawback under the strict regulatory demands imposed by current good manufacturing practice (cGMP). However, we have achieved transient expression of the monoclonal antibody 2G12 and the fluorescent marker protein DsRed in tobacco leaves with ～15% intra‐batch coefficients of variation, which is within the range reported for transgenic plants. We developed models for the transient expression of both proteins that predicted quantitative expression levels based on five parameters: The OD_600nm of Agrobacterium tumefaciens (from 0.13 to 2.00), post‐inoculation incubation temperature (15–30°C), plant age (harvest at 40 or 47 days after seeding), leaf age, and position within the leaf. The expression models were combined with a model of plant biomass distribution and extraction, generating a yield model for each target protein that could predict the amount of protein in specific leaf parts, individual leaves, groups of leaves, and whole plants. When the yield model was combined with a cost function for the production process, we were able to perform calculations to optimize process time, yield, or downstream costs. We illustrate this procedure by transferring the cost function from a production process using transgenic plants to a hypothetical process for the transient expression of 2G12. Our models allow the economic evaluation of new plant‐based production processes and provide greater insight into the parameters that affect transient protein expression in plants. Biotechnol. Bioeng. 2012; 109: 2575–2588. © 2012 Wiley Periodicals, Inc.