Global Analysis of the HrpL Regulon in the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 Reveals New Regulon Members with Diverse Functions

The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a promoter sequence, often designated as the “hrp promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. PSPTO_5633 is the only new HrpL regulon member that is potentially an effector and is now designated HopBM1. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or ΔhopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.     

Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.     

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.     

Reactivation of insertionally inactivated Shiga toxin 2 genes of Escherichia coli O157:H7 caused by nonreplicative transposition of the insertion sequence

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coli O157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5' and 3' ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.     

AlgW regulates multiple Pseudomonas syringae virulence strategies

Gram-negative bacterial pathogens have evolved a number of virulence-promoting strategies including the production of extracellular polysaccharides such as alginate and the injection of effector proteins into host cells. The induction of these virulence mechanisms can be associated with concomitant downregulation of the abundance of proteins that trigger the host immune system, such as bacterial flagellin. In Pseudomonas syringae, we observed that bacterial motility and the abundance of flagellin were significantly reduced under conditions that induce the type III secretion system. To identify genes involved in this negative regulation, we conducted a forward genetic screen with P. syringae pv. maculicola ES4326 using motility as a screening phenotype. We identified the periplasmic protease AlgW as a key negative regulator of flagellin abundance that also positively regulates alginate biosynthesis and the type III secretion system. We also demonstrate that AlgW constitutes a major virulence determinant of P. syringae required to dampen plant immune responses. Our findings support the conclusion that P. syringae co-ordinately regulates virulence strategies through AlgW in order to effectively suppress host immunity.     

GacA directly regulates expression of several virulence genes in Pseudomonas syringae pv. tabaci 11528

A two-component system comprising GacS and GacA affects a large number of traits in many Gram-negative bacteria. However, the signals to which GacS responds, the regulation mechanism for GacA expression, and the genes GacA controls are not yet clear. In this study, several phenotypic tests and tobacco-leaf pathogenicity assays were conducted using a gacA deletion mutant strain (BL473) of Pseudomonas syringae pv. tabaci 11528. To determine the regulation mechanism for gacA gene expression and to identify GacA-regulated genes, we conducted quantitative RT-PCR and electrophoretic mobility shift assay (EMSA) experiments. The results indicated that virulence traits related to the pathogenesis of P. syringae pv. tabaci 11528 are regulated coordinately by GacA and iron availability. They also revealed that several systems coordinately regulate gacA gene expression in response to iron concentration and bacterial cell density and that GacA and iron together control the expression of several virulence genes. EMSA results provided genetic and molecular evidence for direct control of virulence genes by GacA.     

The X-ray crystallographic structure and activity analysis of a Pseudomonas-specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.     

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.     

Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA-->CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gln to Arg.     

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.     

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10(-5) and 1.1×10(-6), depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.     

Characterization of Plasmids Encoding the Phytotoxin Coronatine in Pseudomonas syringae

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.     

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.     

Identification of a novel Pseudomonas syringae Psy61 effector with virulence and avirulence functions by a HrpL-dependent promoter-trap assay

The hrp pathogenicity island of Pseudomonas syringae encodes a type III secretion system (TTSS) that translocates effectors into plant cells. Most genes encoding effectors are dispersed in the P. syringae genome. Regardless of location, all are regulated coordinately by the alternative sigma factor HrpL. An HrpL-dependent promoter-trap assay was developed to screen genomic libraries of P. syringae strains for promoters whose activity in Escherichia coli is dependent on an inducible hrpL construct. Twenty-two HrpL-dependent promoter fragments were isolated from P. syringae Psy61 that included promoters for known HrpL-dependent genes. One fragment also was isolated that shared no similarity with known genes but retained a near consensus HrpL-dependent promoter. The sequence of the region revealed a 375-amino acid open reading frame encoding a 40.5-kDa product that was designated HopPsyL. HopPsyL was structurally similar to other secreted effectors and carried a putative chloroplast-targeting signal and two predicted transmembrane domains. HopPsyL':'AvrRpt2 fusions were translocated into host cells via the P. syringae pv. tomato DC3000 hrp TTSS. A hopPsyL::kan mutant of Psy61 exhibited strongly reduced virulence in Phaseolus vulgaris cv. Kentucky Wonder, but did not appear to act as a defense response suppressor. The ectopically expressed gene reduced the virulence of Pseudomonas syringae DC3000 transformants in Arabidopsis thaliana Col-0. The gene was shown to be conserved in 6 of 10 P. syringae pv. syringae strains but was not detected in 35 strains of other pathovars. HopPsyL appears to be a novel TTSS-dependent effector that functions as a host-species-specific virulence factor in Psy61.     

An extracellular aspartic protease functions in Arabidopsis disease resistance signaling

We have used activation tagging with T-DNA carrying cauliflower mosaic virus 35S enhancers to investigate the complex signaling networks underlying disease resistance in Arabidopsis. From a screen of approximately 5000 lines, we identified constitutive disease resistance (CDR1) encoding an apoplastic aspartic protease, the overexpression of which causes dwarfing and resistance to virulent Pseudomonas syringae. These phenotypes reflect salicylic-acid-dependent activation of micro-oxidative bursts and various defense-related genes. Antisense CDR1 plants were compromised for resistance to avirulent P. syringae and more susceptible to virulent strains than wild type. CDR1 accumulates in intercellular fluid in response to pathogen attacks. Induction of CDR1 generates a small mobile signal, and CDR1 action is blocked by the protease inhibitor pepstatin and by mutations in the protease active sites. We propose that CDR1 mediates a peptide signal system involved in the activation of inducible resistance mechanisms.