Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis thaliana auxin physiology

The Pseudomonas syringae type III effector AvrRpt2 promotes bacterial virulence on Arabidopsis thaliana plants lacking a functional RPS2 gene (rps2 mutant plants). To investigate the mechanisms underlying the virulence activity of AvrRpt2, we examined the phenotypes of transgenic A. thaliana rps2 seedlings constitutively expressing AvrRpt2. These seedlings exhibited phenotypes reminiscent of A. thaliana mutants with altered auxin physiology, including longer primary roots, increased number of lateral roots, and increased sensitivity to exogenous auxin. They also had increased levels of free indole acetic acid (IAA). The presence of AvrRpt2 also was correlated with a further increase in free IAA levels during infection with P. syringae pv. tomato strain DC3000 (PstDC3000). These results indicate that AvrRpt2 alters A. thaliana auxin physiology. Application of the auxin analog 1-naphthaleneacetic acid promoted disease symptom development in PstDC3000-infected plants, suggesting that elevated auxin levels within host tissue promote PstDC3000 virulence. Thus, AvrRpt2 may be among the virulence factors of P. syringae that modulate host auxin physiology to promote disease.     

Pseudomonas syringae Hrp type III secretion system and effector proteins

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNA(Leu) gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.     

A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae

Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.     

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2_Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2_Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2_Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2_Pto. In support of this hypothesis, HopF2_Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2_Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2_Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.     

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a

Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1–resistance complex, resulting in ETI activation.The plant immune system can be divided into two major branches that share commonalities with animal innate immunity. Pattern recognition receptor (PRR)-triggered immunity (PTI) is activated by the recognition of conserved microbial molecules called microbe-associated molecular patterns (MAMPs) by extracellular PRRs (1), similar to the recognition of MAMPs by animal Toll-like receptors (2). Effector-triggered immunity (ETI) is activated by the recognition of pathogen-derived effector proteins by intracellular NB-LRR proteins that share structural features with animal nucleotide-binding domain, leucine-rich repeat containing (NLR) proteins (3). The ETI response overlaps significantly with PTI but is accelerated, amplified, and often accompanied by the hypersensitive cell death response (HR; refs. 4 and 5). Recognition of effector proteins can occur directly where the effector protein binds directly to the NB-LRR protein, or indirectly, in which case both the effector and NB-LRR proteins bind to a common host protein. In the latter case, ETI is initiated by the NB-LRR protein in response to an effector-induced modification to the host protein (6). In some cases, the host protein monitored by the NB-LRR may represent a true virulence target of the effector protein, whereas in other cases, this protein may be a nonfunctional decoy of the true virulence target maintained by the host for the purposes of pathogen surveillance (7).The continual arms race between host and pathogen has directed the coevolution of host innate immunity with bacterial virulence strategies. The type III secretion system (T3SS) is a predominant virulence mechanism used by many Gram-negative bacterial pathogens to cause disease in eukaryotic hosts (8). The T3SS delivers type III secreted effector (T3SE) proteins into host cells where they can promote the infection process by suppressing host immunity and/or participating in the pathogen life cycle.The YopJ/HopZ/AvrRxv T3SE superfamily is evolutionary diverse and found in both animal and plant pathogens (9, 10). Yersinia pestis YopJ, the archetypal member of this superfamily, acetylates serine or threonine residues in the activation loops of members of the mitogen-activated protein kinase kinase (MAPKK) and MAP kinase kinase kinase superfamilies, thereby suppressing innate immunity (11–15). In the plant pathogen Pseudomonas syringae, the HopZ1 subfamily is represented by three closely related alleles, HopZ1a, HopZ1b, and HopZ1c, that diversified under pressure from the host immune system (10). The most phylogenetically basal representative of this subfamily, HopZ1a, is recognized in Arabidopsis by the ZAR1 NB-LRR protein (16, 17) as well as by unidentified proteins in rice, Nicotiana benthamiana, sesame, and soybean (10). Like YopJ, HopZ1a is an acetyltransferase and can promote pathogen growth in Arabidopsis plants lacking the ZAR1 NB-LRR protein. The virulence and immune-eliciting functions of HopZ1a both require the cysteine residue in the acetyltransferase catalytic triad (16, 17). It is likely that ZAR1 evolved to recognize an ancestral virulence activity of HopZ1a; however, the molecular relationship between virulence and immunity-eliciting functions remain to be established (17, 18).Here, we describe a forward genetic screen designed to characterize the genetic requirements of ZAR1-mediated immunity by identifying Arabidopsis mutant plants that lacked a HopZ1a-induced HR response. We named these mutants hopZ-ETI deficient (zed) and mapped zed1 to At3g57750 by Illumina-based next-generation sequencing. Different zed1 point mutants identified from our mutant screen or a tDNA insertion line in At3g57750 all lack recognition of HopZ1a. PTI and basal defenses are unaffected in zed1; however, zed1 exhibits a loss of HopZ1a-mediated ETI. We show that ZED1 interacts directly with HopZ1a, as well as the N-terminal coiled-coil (CC) domain of ZAR1. ZED1 is an uncharacterized pseudokinase and is acetylated on threonines 125 and 177 by HopZ1a. We hypothesize that ZAR1 is activated in response to ZED1 acetylation by HopZ1a and speculate that ZED1 may be a decoy of the true HopZ1a virulence target because HopZ1a retains its virulence function in zed1 Arabidopsis plants.     

Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host–pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.Plants evoke resistance against invading bacteria using plasma membrane-localized pattern recognition receptors (PRRs) to detect the presence of pathogen-associated molecular patterns (PAMPs) in the extracellular space (1). Activation of PRRs by PAMPs results in numerous defense responses that limit bacterial growth (1). However, the actual mechanisms by which plants suppress virulence and restrict bacterial growth remain unclear. Pseudomonas syringae is a model bacterial pathogen that infects a wide range of economically important crops as well as the laboratory model plant Arabidopsis (2). P. syringae uses several different virulence strategies to suppress host defenses, including a type III secretion system (T3SS) that secretes up to 30 effector proteins into plant cells (3, 4). Many effectors function to suppress PRR-induced signaling, thereby allowing the bacteria to avoid detection and proliferate (4). Mutants of P. syringae lacking a functional T3SS are not fully virulent, demonstrating that this system is essential for a successful infection (5, 6). Moreover, recent studies have revealed that PAMP-triggered immunity (PTI) leads to a restriction in the delivery of type III effectors into host cells, suggesting that plants possess an unknown mechanism(s) to block type III secretion (7, 8).Despite the critical role of the T3SS in P. syringae virulence, T3SS structural components and effectors are not constitutively present but are produced at the onset of infection (9, 10). Early attempts to identify plant signals perceived by P. syringae revealed that synthetic medium mimicking the plant apoplast, namely a minimal nutrient medium with acidic pH and including a sugar such as fructose, is capable of inducing T3SS-associated genes (9–12). However, in some instances expression of the T3SS was higher in planta than in synthetic medium, indicating that additional plant-derived factors likely were required for full induction (10, 12). These results imply the presence of plant-derived signal(s) that induce the T3SS, and various signals have been proposed to be capable of inducing the T3SS in different plant pathogenic bacteria based largely on in vitro experiments (10, 12–17). However, whether any of these signals affect the biological outcome of the host–pathogen interaction remains speculative because of the lack of genetic mutants altering the abundance of these chemical signals in the host.In the present work, we identify host chemical signals that DC3000 uses to switch to its virulence program and demonstrate that this recognition event plays an important role in a successful infection. The identification of an Arabidopsis mutant, MAPK phosphatase 1 (mkp1), in which the delivery of the P. syringae pv tomato DC3000 effector is suppressed, provided an important genetic model for investigating the basis for T3SS induction. Using a metabolomics comparison of mutant and WT plant exudates, we identified several plant-derived metabolites that are present at lower levels in mkp1 and induce the T3SS in DC3000. The biological significance of these compounds was demonstrated by showing that reintroducing these T3SS-inducing metabolites can overcome both the suppression of effector delivery and the enhanced resistance in mkp1 plants. Furthermore, the addition of these metabolites also can overcome enhanced resistance induced in plants pretreated with PAMPs. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the levels of these host-derived signals contribute to susceptibility or resistance.     

Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field

hrp genes are reportedly required for pathogenicity in Pseudomonas syringae pv. syringae (Pss) and other phytopathogenic bacterial species. A subset of these genes encodes a type III secretion system through which virulence factors are thought to be delivered to plant cells. In this study, we sought to better understand the role that hrp genes play in interactions of Pss with its host as they occur naturally under field conditions. Population sizes of hrp mutants with defects in genes that encode components of the Hrp secretion system (DeltahrcC::nptII and hrpJ:: OmegaSpc) and a protein secreted via the system (DeltahrpZ::nptII) were similar to B728a on germinating seeds. However, phyllosphere (i.e., leaf) population sizes of the hrcC and hrpJ secretion mutants, but not the hrpZ mutant, were significantly reduced relative to B728a. Thus, the Hrp type III secretion system, but not HrpZ, plays an important role in enabling Pss to flourish in the phyllosphere, but not the spermosphere. The hrcC and hrpJ mutants caused brown spot lesions on primary leaves at a low frequency when they were inoculated onto seeds at the time of planting. Pathogenic reactions also were found when the hrp secretion mutants were co-infiltrated into bean leaves with a non-lesion-forming gacS mutant of B728a. In both cases, the occurrence of disease was associated with elevated population sizes of the hrp secretion mutants. The role of the Hrp type III secretion system in pathogenicity appears to be largely mediated by its requirement for growth of Pss in the phyllosphere. Without growth, disease does not occur.     

The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants

The plant pathogenic bacterium Pseudomonas syringae is divided into pathovars differing in host specificity, with P. syringae pv. syringae (Psy) and P. syringae pv. tomato (Pto) representing particularly divergent pathovars. P. syringae hrp/hrc genes encode a type III protein secretion system that appears to translocate Avr and Hop effector proteins into plant cells. DNA sequence analysis of the hrp/hrc regions in Psy 61, Psy B728a, and Pto DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The hrp/hrc gene cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EELs begin 3 nt downstream of the stop codon of hrpK and end, after 2.5-7.3 kb of dissimilar intervening DNA with tRNA(Leu)-queA-tgt sequences that are also found in Pseudomonas aeruginosa but without linkage to any Hrp Pai sequences. The EELs encode diverse putative effectors, including HopPsyA (HrmA) in Psy 61 and proteins similar to AvrPphE and the AvrB/AvrC/AvrPphC and AvrBsT/AvrRxv/YopJ protein families in Psy B728a. The EELs also contain mobile genetic element sequences and have a G + C content significantly lower than the rest of the Hrp Pai or the P. syringae genome. The CEL carries at least seven ORFs that are conserved between Psy B728a and Pto DC3000. Deletion of the Pto DC3000 EEL slightly reduces bacterial growth in tomato, whereas deletion of a large portion of the CEL strongly reduces growth and abolishes pathogenicity in tomato.     

Elucidation of a pH-folding switch in the Pseudomonas syringae effector protein AvrPto

Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly conserved type III secretion system (T3SS) provides a regulated conduit between the bacterial and host cytoplasm for delivery of a specific set of bacterial effector proteins that serve to disrupt host signaling and metabolism for the benefit of the bacterium. Remarkably, the inner diameter of the T3SS apparatus requires that effector proteins pass through in at least a partially unfolded form. AvrPto, an effector protein of the plant pathogen Pseudomonas syringae, adopts a helical bundle fold of low stability (ΔG^F→U = 2 kcal/mol at pH 7, 26.6 °C) and offers a model system for chaperone-independent secretion. P. syringae effector proteins encounter a pH gradient as they translocate from the bacterial cytoplasm (mildly acidic) into the host cell (neutral). Here, we demonstrate that AvrPto possesses a pH-sensitive folding switch controlled by conserved residue H87 that operates precisely in the pH range expected between the bacterial and host cytoplasm environments. These results provide a mechanism for how a bacterial effector protein employs an intrinsic pH sensor to unfold for translocation via the T3SS and refold once in the host cytoplasm and provide fundamental insights for developing strategies for delivery of engineered therapeutic proteins to target tissues.     

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae

The virulence of Pseudomonas syringae and many other proteobacterial pathogens is dependent on complex repertoires of effector proteins injected into host cells by type III secretion systems. The 28 well-expressed effector genes in the repertoire of the model pathogen P. syringae pv. tomato DC3000 were deleted to produce polymutant DC3000D28E. Growth of DC3000D28E in Nicotiana benthamiana was symptomless and 4 logs lower than that of DC3000ΔhopQ1-1, which causes disease in this model plant. DC3000D28E seemed functionally effectorless but otherwise WT in diagnostic phenotypes relevant to plant interactions (for example, ability to inject the AvrPto-Cya reporter into N. benthamiana). Various effector genes were integrated by homologous recombination into native loci or by a programmable or random in vivo assembly shuttle (PRIVAS) system into the exchangeable effector locus in the Hrp pathogenicity island of DC3000D28E. The latter method exploited dual adapters and recombination in yeast for efficient assembly of PCR products into programmed or random combinations of multiple effector genes. Native and PRIVAS-mediated integrations were combined to identify a minimal functional repertoire of eight effector genes that restored much of the virulence of DC3000ΔhopQ1-1 in N. benthamiana, revealing a hierarchy in effector function: AvrPtoB acts with priority in suppressing immunity, enabling other effectors to promote further growth (HopM1 and HopE1), chlorosis (HopG1), lesion formation (HopAM1-1), and near full growth and symptom production (AvrE, HopAA1-1, and/or HopN1 functioning synergistically with the previous effectors). DC3000D28E, the PRIVAS method, and minimal functional repertoires provide new resources for probing the plant immune system.     

The Pseudomonas syringae effector AvrRpt2 cleaves its C-terminally acylated target, RIN4, from Arabidopsis membranes to block RPM1 activation

Plant pathogenic Pseudomonas syringae deliver type III effector proteins into the host cell, where they function to manipulate host defense and metabolism to benefit the extracellular bacterial colony. The activity of these virulence factors can be monitored by plant disease resistance proteins deployed to "guard" the targeted host proteins. The Arabidopsis RIN4 protein is targeted by three different type III effectors. Specific manipulation of RIN4 by each of them leads to activation of either the RPM1 or RPS2 disease resistance proteins. The type III effector AvrRpt2 is a cysteine protease that is autoprocessed inside the host cell where it activates RPS2 by causing RIN4 disappearance. RIN4 contains two sites related to the AvrRpt2 cleavage site (RCS1 and RCS2). We demonstrate that AvrRpt2-dependent cleavage of RIN4 at RCS2 is functionally critical in vivo. This event leads to proteasome-mediated elimination of all but a membrane-embedded approximately 6.4-kDa C-terminal fragment of RIN4. One or more of three consecutive cysteines in this C-terminal fragment are required for RIN4 localization; these are likely to be palmitoylation and/or prenylation sites. AvrRpt2-dependent cleavage at RCS2, and release of the remainder of RIN4 from the membrane, consequently prevents RPM1 activation by AvrRpm1 or AvrB. RCS2 is contained within the smallest tested fragment of RIN4 that binds AvrB in vitro. Thus, at least two bacterial virulence factors target the same domain of RIN4, a approximately 30-aa plant-specific signature sequence found in a small Arabidopsis protein family that may be additional targets for these bacterial virulence factors.     

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a co-chaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.     

Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa

The homologues LcrV of Yersinia species and PcrV of Pseudomonas aeruginosa are pore-forming components. When expressed in a Yersinia lcrV background, PcrV formed smaller pores in infected erythrocyte membranes, correlating to a lowered translocation of Yersinia effectors. To understand this phenomenon, cytotoxins exoenzyme S of P. aeruginosa and YopE of Yersinia were introduced into a Yersinia background without Yop effectors but expressing LcrV or PcrV. Comparable translocation of each substrate indicated that substrate recognition by LcrV/PcrV is not a regulator of translocation. Yersinia harboring pcrV coexpressed with its native operon efficiently translocated effectors into HeLa cell monolayers and formed large LcrV-like pores in erythrocyte membranes. Thus, a PcrV complex with native P. aeruginosa translocon components is required to form fully functional pores for complete complementation of effector translocation in Yersinia.     

Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000

The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.     

铜绿假单胞菌Ⅲ型分泌系统的研究进展

铜绿假单胞菌是临床常见的重要条件致病菌,具有多种毒力因子,能引起各种急慢性感染。其中最重要的毒力因子是Ⅲ型分泌系统,主动向宿主细胞靶向输送效应蛋白,引起宿主细胞的病理变化,并逃避免疫细胞的降解。研究Ⅲ型分泌系统不仅有助于明确铜绿假单胞菌的致病机制,更重要的是为临床治疗及新药研发提供新思路。     

Melatonin regulates carbohydrate metabolism and defenses against Pseudomonas syringae pv. tomato DC3000 infection in Arabidopsis thaliana

Melatonin has been reported to promote plant growth and development. Our experiments with Arabidopsis thaliana showed that exogenous applications of this molecule mediated invertase inhibitor (C/VIF)‐regulated invertase activity and enhanced sucrose metabolism. Hexoses were accumulated in response to elevated activities by cell wall invertase (CWI) and vacuolar invertase (VI). Analyses of sugar metabolism‐related genes revealed differential expression during plant development that was modulated by melatonin. In particular, C/VIF1 and C/VIF2 were strongly down‐regulated by exogenous feeding. We also found the elevated CWI activity in melatonin‐treated Arabidopsis improved the factors (cellulose, xylose, and galactose) for cell wall reinforcement and callose deposition during Pseudomonas syringae pv. tomato DC3000 infection, therefore, partially induced the pathogen resistance. However, CWI did not involve in salicylic acid (SA)‐regulated defense pathway. Taken together, this study reveals that melatonin plays an important role in invertase‐related carbohydrate metabolism, plant growth, and pathogen defense.     

Detection of Pseudomonas aeruginosa type III antibodies in children with tracheostomies

Pseudomonas aeruginosa is often cultured from the airways of children with tracheostomies. P. aeruginosa produces exotoxin A (ETA) and type III cytotoxins. This study tested the hypothesis that children with tracheostomies are colonized by P. aeruginosa that express these virulence factors and will have antibodies directed against these virulence factors, indicating infection rather than only colonization. A convenience sample of 30 patients, ranging in age from 2 months-22 years, was recruited. Serum was tested for the presence of antibodies to ETA and components of the type III system by Western blot analysis. Twenty-one of 39 patients (70%) had antibodies to components of the type III system. Fifteen of 30 (50%) were seropositive for ETA. Sera from patients who were antibody-positive for ETA were also seropositive for either ExoS or ExoU. Nine of 30 patients (30%) did not possess antibodies to ETA or components of the type III system. In conclusion, these data identified a seropositive reaction to P. aeruginosa cytotoxins in some patients with tracheostomies, suggestive of infection by cytotoxic strains of P. aeruginosa. Future studies will determine the utility of measuring seroconversion to these cytotoxins as an early indication of infection in children with tracheostomies.