The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as-yet-unidentified avirulence factor possessed by the Japanese tobacco-avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector-triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence-related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid-resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco-virulent strain BK1002 contains mutations in ripB and expresses a C-terminal-truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine-tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB-induced resistance against R. solanacearum was abolished in Roq1-silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.     

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.     

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.     

细菌三型分泌系统效应蛋白转运的研究进展

三型分泌系统(Type 3 secretion system,T3SS)作为存在于革兰氏阴性菌中的分泌系统之一,对革兰氏阴性菌的致病有重要作用。T3SS的致病作用体现在T3SS能直接将效应蛋白转运至宿主细胞,进而通过效应蛋白调控细胞的一系列通路,促进细菌定殖于细胞。而效应蛋白的转运受到两方面因素的调控,一方面是效应蛋白本身的信号序列,另一方面是T3SS相关蛋白的辅助。本文围绕近年来T3SS的构成、效应蛋白转运机制方面的最新进展进行概要综述。     

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.     

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.     

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.     

Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine‐learning approach

The Gram‐negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85‐10 strain by various experimental and computational techniques. To identify additional Xcv 85‐10 effectors, we applied a genome‐wide machine‐learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)‐dependent expression, 5′ regulatory sequences, amino acid composition bias and GC content. Thirty‐six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)‐inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild‐type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild‐type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.     

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 NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT.     

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.     

铜绿假单胞菌中III型分泌系统受Rhl和PQS群体感应系统调节

摘要：【目的】研究铜绿假单胞菌中群体感应系统（Quorum sensing, QS）与III型分泌系统（Type III secretion system, T3SS）的关系。【方法】通过基因敲除的方法破坏铜绿假单胞菌QS系统相关基因,将T3SS相关基因exoS、exoY、exoT、exsD-pscA-L启动子-报道子luxCDABE融合体整合到野生型菌株及QS系统突变菌株的染色体组上,通过检测启动子活性,比较这些基因在不同菌株中的表达情况。【结果】研究结果表明,T3SS中的exoS与exoT在pqsR基因突变体中的表达有明显的增强,Rhl系统对这四种基因的表达具有抑制作用,而Las系统存在与否对T3SS基本没有影响。【结论】铜绿假单胞菌中的Rhl系统和奎诺酮信号（Pseudomonas Quinolone Signal, PQS）系统对T3SS相关基因的表达具有重要的调节作用。     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.     

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.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

铜绿假单胞菌中Ⅲ型分泌系统调控机制及针对性治疗的研究进展

铜绿假单胞菌是临床上重要的条件致病菌,具有多种毒力因子且极易产生耐药性。Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)是铜绿假单胞菌中重要的毒性因子分泌系统,该菌通过Ⅲ型分泌系统将多种毒力因子注入到真核宿主细胞内并逃逸宿主细胞免疫系统的清除,引起宿主细胞相应的病理变化。对Ⅲ型分泌系统的研究,不仅有助于明确铜绿假单胞菌的致病机理,更可为临床治疗和药物研发提供理论基础。本文主要对铜绿假单胞菌中Ⅲ型分泌系统的结构、功能、调控机制以及针对性治疗策略等方面的研究进行了综述。     

Evaluation of the taxonomic accuracy and pathogenicity prediction power of 16 primer sets amplifying single copy marker genes in the Pseudomonas syringae species complex

The Pseudomonas syringae species complex is composed of several closely related species of bacterial plant pathogens. Here, we used in silico methods to assess 16 PCR primer sets designed for broad identification of isolates throughout the species complex. We evaluated their in silico amplification rate in 2161 publicly available genomes, the correlation between pairwise amplicon sequence distance and whole genome average nucleotide identity, and trained naive Bayes classification models to quantify classification resolution. Furthermore, we show the potential for using single amplicon sequence data to predict type III effector protein repertoires, which are important determinants of host specificity and range.     

Inhibition of the type III secretion system by syringaldehyde protects mice from Salmonella enterica serovar Typhimurium

The invasiveness of Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely associated with the Salmonella pathogenicity island (SPI)‐encoded type Ⅲ secretion system (T3SS), which can directly inject a series of effector proteins into eukaryotic cells to enable bacterial infection. In this study, syringaldehyde was identified as an effective inhibitor of the S. Typhimurium T3SS using an effector protein‐lactamase fusion reporter system. Syringaldehyde treatment could inhibit the expression of important effector proteins (SipA, SipB and SipC) at a concentration of 0.18 mM without affecting bacterial growth. Additionally, significant inhibition of bacterial invasion and cellular injury was observed following the syringaldehyde treatment in the co‐infection system of HeLa cells and S. Typhimurium. Furthermore, treatment with syringaldehyde provided systemic protection to mice infected with S. Typhimurium, reducing mortality (40.00%) and bacterial loads and relieving caecal damage and systemic inflammation. The results presented in this study indicate that syringaldehyde significantly affects T3SS activity and is a potential leading compound for treating S. Typhimurium infections.