The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.     

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.     

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.     

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.     

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.     

Hrp pilus: An hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6–8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.     

Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean

Three different cosmid clones were isolated from a genomic library of the tomato pathogen Pseudomonas syringae pv. tomato, which, when introduced into the soybean pathogen P. syringae pv. glycinea, caused a defensive hypersensitive response (HR) in certain soybean cultivars. Each clone was distinguished by the specific cultivars that reacted hypersensitively and by the intensity of the HR elicited. Unlike wild-type P. syringae pv. tomato isolates, which elicit the HR on all soybean cultivars, all three clones exhibited cultivar specificities analogous to avirulence genes previously cloned from P. syringae pv. glycinea. However, the collective phenotypes of the three clones accounted for HRs on all tested soybean cultivars. One of the three P. syringae pv. tomato clones contained an avirulence gene homologous to avrA, which was previously cloned from P. syringae pv. glycinea race 6. The other two P. syringae pv. tomato clones expressed unique HR patterns on various soybean cultivars, which were unlike those caused by any known P. syringae pv. glycinea race or previously cloned P. syringae pv. glycinea avr gene. Further characterization of the second P. syringae pv. tomato clone indicated that the avirulence phenotype resided on a 5.6-kilobase HindIII fragment that, in Southern blot analyses, hybridized to an identical-size fragment in various P. syringae pathovars, including all tested glycinea races. These results demonstrate that avirulence genes may be distributed among several P. syringae pathovars but may be modified so that the HR is not elicited in a particular host plant. Furthermore, the data raise the possibility that avirulence genes may function in host-range determination at levels above race—cultivar specificity.     

The gene coding for the Hrp pilus structural protein is required for type III secretion of Hrp and Avr proteins in Pseudomonas syringae pv. tomato

Bacterial surface appendages called pili often are associated with DNA and/or protein transfer between cells. The exact function of pili in the transfer process is not understood and is a matter of considerable debate. The Hrp pilus is assembled by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato (Pst) strain DC3000. In this study, we show that the hrpA gene, which encodes the major subunit of the Hrp pilus, is required for secretion of putative virulence proteins, such as HrpW and AvrPto. In addition, the hrpA gene is required for full expression of genes that encode regulatory, secretion, and effector proteins of the type III secretion system. hrpA-mediated gene regulation apparently is through effect on the mRNA level of two previously characterized regulatory genes, hrpR and hrpS. Ectopic expression of the hrpRS gene operon restored gene expression, but not protein secretion, in the hrpA mutant. Three single amino acid mutations at the HrpA carboxyl terminus were identified that affect the secretion or regulatory function of the HrpA protein. These results define an essential role of the Hrp pilus structural gene in protein secretion and coordinate regulation of the type III secretion system in Pst DC3000.     

Genome,Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.     

Biological and Molecular Characterization of the Lytic Bacteriophage SoKa against Pseudomonas syringae pv. syringae,Causal Agent of Citrus Blast and Black Pit in Tunisia

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between −80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol.     

Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L.) Merr

A genomic library of Pseudomonas syringae pv. glycinea race 6 DNA was constructed in the mobilizable cosmid vector pLAFR1 and maintained in Escherichia coli HB101. Completeness of the library was estimated by assaying clones for the expression of ice-nucleating activity in E. coli. Ice-nucleation activity was represented approximately once in every 600 clones. Six hundred eighty random race 6 cosmid clones were mobilized from E. coli by plasmid pRK2013 in individual conjugations to a race 5 strain of P. s. glycinea. A single clone (pPg6L3) was detected that changed the race specificity of race 5 from virulent (compatible) to avirulent (incompatible) on the appropriate soybean cultivars. The clone was also mobilized from E. coli into race 1 and race 4 strains of P. s. glycinea, and it conferred on these transconjugants the same host range incompatibility as the wild-type race 6 strain. The cosmid clone was mapped by restriction endonucleases, and two adjacent EcoRI fragments were identified by transposon Tn5 mutagenesis to be important in determining race specificity. Southern blot analysis showed that the two EcoRI fragments are unique to race 6 and are not present in the other races tested. The cosmid clone pPg6L3 was also mobilized to Pseudomonas fluorescens and Rhizobium japonicum. However, neither these isolates nor E. coli harboring pPg6L3 elicited a hypersensitive reaction in soybean leaves.     

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 °C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 °C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.     

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.     

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.     

Plant intracellular innate immune receptor Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) is activated at, and functions on, the plasma membrane

Plants deploy intracellular innate immune receptors to recognize pathogens and initiate disease resistance. These nucleotide-binding, leucine-rich repeat (NB-LRR) proteins are activated by pathogen effector proteins that are delivered into the host cell to suppress host defense responses. Little is known about the sites and mechanisms of NB-LRR activation, but some NB-LRR proteins can function inside the plant nucleus. We demonstrate that RPM1 is activated on the plasma membrane and does not relocalize to the nucleus. An autoactive RPM1(D505V) allele that recapitulates key features of normal RPM1 activation also resides on the plasma membrane. There is no detectable relocalization of activated RPM1 to the nucleus. Hindering potential nuclear entry of RPM1-Myc did not affect either its effector-triggered hypersensitive-response (HR) cell death or its disease resistance functions, further suggesting that nuclear translocation is not required for RPM1 function. RPM1 tethered onto the plasma membrane with a dual acylated N-terminal epitope tag retained the ability to mediate HR, consistent with this RPM1 function being activated on the plasma membrane. Plant NB-LRR proteins can thus function at various locations in the cell.     

Comparison of complete sequences of hepatitis B virus genotype C between inactive carriers and hepatocellular carcinoma patients before and after seroconversion

Background Most patients who acquire chronic hepatitis B virus (HBV) infection by perinatal transmission become inactive carriers (IC) after hepatitis B e (HBe) antigen seroconversion, whereas some patients have persistent abnormal serum transaminase levels and develop hepatocellular carcinoma (HCC) in the anti-HBe-positive phase. The aim of this study was to investigate the HCC-related mutations of HBV. Methods Complete sequences of HBV were examined among eight IC and eight HCC patients infected with HBV genotype C before and after seroconversion. Results The frequency of the T1653 mutation tended to be higher among HCC patients after seroconversion (16.7% vs. 62.5%; P = 0.086). The prevalence of a basal core promoter double mutation (T1762/A1764) was high among both IC and HCC patients after seroconversion (83.3% vs. 87.5%; P = 0.825). Among the HCC patients, a pre-S deletion mutant was detected in 62.5% patients before seroconversion, and in 37.5% patients after seroconversion. The core deletion mutant was also detected in 50% of HCC patients only before seroconversion. Deletion mutants of the pre-S or core region before seroconversion were significantly associated with HCC patients (0% vs. 62.5%; P = 0.007, 0% vs. 50%; P = 0.021, respectively). Conclusions Our data showed a significant association of pre-S and core deletion mutants before seroconversion with HCC development. The T1653 mutation after seroconversion was frequently found in HCC patients infected with HBV genotype C. These results suggest that mutations may be predictive factor for development of HCC.