Suvi Taira

Genomic Organization of Human and Mouse Genes for Vascular Endothelial Growth Factor C

We report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-κB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family.

An efficient and accurate integration of mini-Mu transposons in vitro : a general methodology for functional genetic analysis and molecular biology applications

Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins.

The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ

The type III secretion system (TTSS) is an essential requirement for the virulence of many Gram‐negative bacteria infecting plants, animals and man. Pathogens use the TTSS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cell, where the effectors subvert host defences. Plant pathogens have to translocate their effector proteins through the plant cell wall barrier. The best candidates for directing effector protein traffic are bacterial appendages attached to the membrane‐bound components of the TTSS. We have investigated the protein secretion route in relation to the TTSS appendage, termed the Hrp pilus, of the plant pathogen Pseudomonas syringae pv. tomato. By pulse expression of proteins combined with immunoelectron microscopy, we show that the Hrp pilus elongates by the addition of HrpA pilin subunits at the distal end, and that the effector protein HrpZ is secreted only from the pilus tip. Our results indicate that both HrpA and HrpZ travel through the Hrp pilus, which functions as a conduit for the long‐distance translocation of effector proteins.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Type III Secretion Contributes to the Pathogenesis of the Soft-Rot Pathogen Erwinia carotovora: Partial Characterization of the hrp Gene Cluster

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

MUTATIONAL ANALYSIS OF THE PSEUDOMONAS SYRINGAE PV. TOMATO HRPA GENE ENCODING HRP PILUS SUBUNIT

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus‐dependent autoagglutination of the bacteria. The majority of insertions in the amino‐terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy‐terminus appeared to be needed for pilus assembly. Insertions in the 5′ non‐translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages

The plasmid‐carried spv genes promote virulence of salmonellae in mice by allowing bacterial growth in the reticuloendothelial tissue. When the bacteria are cultivated under normal laboratory conditions the spv genes appear dormant. This communication explores the transcriptional regulation of spv genes within murine macrophage‐like J774‐A.1 cells utilizing a new reporter system. Transcriptional fusions were constructed between promoter elements of the Salmonella enterica var. Typhimurium spv genes and the KS71A fimbrial gene cluster. The expression of KS71A fimbriae In fusion‐carrying Escherichia coli strains was found to be under the control of the transcriptional activator gene spvR. In strains overproducing SpvR, KS71A fimbriae were assembled on the bacterial cell surface and could be detected by bacterial agglutination or immunofluorescence of intact bacteria; the reporter activity was quantified by estimating the percentage of fluorescent bacteria and by immunoblotting of cell lysates. The activity of the reporters, when transformed into the parent Typhimurium TML R66, was low and revealed less than 0.3% fimbriated cells under in vitro culture conditions. A 15–30‐fold increase in fimbriation was observed when the bacteria were cultivated within J774‐A.1 cells. No such increase occurred when the reporter fusions were transformed into TML R66 cured of the virulence plasmid. Insertional inactivation of the spvR gene of the virulence plasmid in Typhimurium TML R66 also abolished induction, whereas corresponding inactivation of spvA or spvB did not reduce induction. No increase in reporter activity was obtained in Typhimurium of line Q1, which is naturally avirulent for mice, although the strain was provided with virulence plasmid pEX102 of line TML R66. We conclude that the intracellular environment of J774‐A.1 cells induces the spv genes and that this induction requires gene functions of both the bacterial chromosome and the virulence plasmid.

Localization of hrpA‐induced Pseudomonas syringae pv. tomato DC3000 in infected tomato leaves

SUMMARY Pseudomonas syringae pv. tomato is the causative agent of bacterial speck of tomato. The key virulence determinant of P. syringae is the hrp gene cluster, which encodes a type III secretion system. The type III system is used by a wide variety of pathogenic bacteria for transporting virulence proteins from the bacteria directly into the eukaryotic host cell. Hrp pilus, which is composed of HrpA pilin subunits, is an indispensable component of the type III secretion system in P. syringae. Here we have determined the spatial and temporal expression pattern of hrpA of P. syringae DC3000 in intact leaves, using a HrpA-GFP protein fusion and confocal microscopy. The hrpA gene was strongly and rapidly induced inside the leaf tissues after infiltration of the bacteria. After spray-inoculation, hrpA-induced bacteria were detected endophytically 72 h post-inoculation, and 96 h after spray-inoculation, disease symptoms appeared and GFP-expressing bacteria were observed at symptom sites, both endo- and epiphytically. Live/dead staining of the bacteria showed that Pst DC3000 does not survive well on leaf surfaces. Apoplastic populations were apparently bursting on to the leaf surface through stomata. Kinetics of population sizes of wild-type DC3000 and hrpA(-) showed significant differences, initially endophytically and only later epiphytically. Our results suggest that the Hrp pilus is first induced in the apoplast and apparently functions mainly inside the leaf tissues. These results suggest that P. syringae DC3000 mainly multiplies endophytically.

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.

Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity

The HrpZ1 gene product from phytopathogenic Pseudomonas syringae is secreted in a type-III secretion system-dependent manner during plant infection. The ability of HrpZ1 to form ion-conducting pores is proposed to contribute to bacterial effector delivery into host cells, or may facilitate the nutrition of bacteria in the apoplast. Furthermore, HrpZ1 is reminiscent of a pathogen-associated molecular pattern (PAMP) that triggers immunity-associated responses in a variety of plants. Here, we provide evidence that the ion pore formation and immune activation activities of HrpZ1 have different structure requirements. All HrpZ1 orthologous proteins tested possess pore formation activities, but some of these proteins fail to trigger plant defense-associated responses. In addition, a C-terminal fragment of HrpZ1 retains the ability to activate plant immunity, whereas ion pore formation requires intact HrpZ1. Random insertion mutagenesis of HrpZ1 further revealed the C terminus to be important for the PAMP activity of the protein. HrpZ1 binds to plant membranes with high affinity and specificity, suggesting that the activation of plant immunity-associated responses by HrpZ1 is receptor-mediated. Our data are consistent with dual roles of HrpZ1 as a virulence factor affecting host membrane integrity, and as a microbial pattern governing the activation of plant immunity during infection.