Hrp Region Research Articles

Alternative Sigma Factor HrpL of Pectobacterium carotovorum 35 is Important for the Development of Soft-rot Symptoms

A bacterial artificial chromosome library of Pectobacterium carotovorum 35 was constructed to characterize the genome and to sequence its hrp region. The hrp cluster of P. carotovorum 35 consisted of 26 open reading frames in five operons. A promoter-based green fluorescent protein technology was used to identify the genes regulated by the alternative sigma factor, HrpL, in P. carotovorum 35. The majority of the selected clones contained the hrpJ operon promoter sequence, which harbors a hrp box, but no putative hrp boxes were detected within the promoter sequences of two other hrpL-regulated genes encoding for pectate lyase and large repetitive protein. Although the promoters of five other hrp operons also contained hrp boxes, their expression was not HrpL-dependent in the promoter-based selection in E. coli. However, transcriptional analysis showed that expression from all operons harboring hrp boxes, except for the hrpN operon, was reduced significantly in the hrpL mutant. The severity of soft-rot symptoms when the hrpL mutant was applied to the surface of tobacco leaves, mimicking natural infection, was greatly attenuated. These results indicate that the hrpL gene of P. carotovorum 35 may be involved in the development of soft-rot symptoms.

Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes

Efficient control of Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, requires a sensitive and reliable diagnostic tool. A PCR detection method that utilizes primers to target the hrp gene cluster region was developed in this study. The nucleotide sequence of the PCR product amplified with primers specific for the hrp region of the xanthomonads and genomic DNA of X. arboricola pv. pruni was determined, and the sequence was aligned with that of X. campestris pv. campestris, which was obtained from the GenBank database. On the basis of the sequence of the amplified hrp region, a PCR primer set of XapF/R specific to X. arboricola pv. pruni was designed. This primer set yielded a 243-bp product from the genomic DNA of X. aboricola pv. pruni strains, but no products from other 21 strains of Xanthomonas or from two epiphytic bacterial species. Southern blot hybridization with the probe derived from the PCR product with the primer set and X. aboricola pv. pruni DNA confirmed the PCR results. The Xap primer system was successfully applied to detect the pathogen from infected peach fruits. When it was applied in field samples, the primer set was proved as a reliable diagnostic tool for specific detection of X. aboricola pv. pruni from peach orchards.

Suppressed surface‐recombination structure and surface passivation for improving current gain of 4H‐SiC BJTs

Abstract4H‐SiC bipolar junction transistors (BJTs) are one of the promising candidates for next‐generation power devices. 4H‐SiC BJTs have the advantages of low on‐resistance and high temperature capability. On the other hand, high common emitter current gain is required from a practical use point of view because BJTs are current‐controlled devices. In order to improve the current gain of 4H‐SiC BJTs, we have concentrated on suppressing surface recombination on the SiC surface, which is a critical limiting factor of the current gain and can be suppressed by reducing carrier density and/or trap density. We have proposed novel 4H‐SiC bipolar junction transistors with suppressed surface‐recombination structure: SSR‐BJTs that have the SiC surface with low carrier density, characterized by a lightly doped n‐type layer (LDN layer) and a highly resistive p‐type region (HRP region). In addition, surface passivation suitable for reducing surface traps has been investigated by applying a new characterization method of surface‐recombination current in which the sp · Ls value, a product of a surface‐recombination velocity (sp) and a surface‐diffusion length (Ls), is derived from an analysis of forward I–V characteristics of SiC pn diodes. We have experimentally demonstrated that the sp · Ls value is a practical indicator to evaluate surface passivation. By using the characterization, an effective passivation method with the combination of a pyrogenic oxidation, a post‐oxidation anneal in H2 ambient, and an anneal in NH3 ambient has been developed. SSR‐BJTs with a variety of device structures and process conditions have been fabricated to investigate their characteristics. A fabricated SSR‐BJT showed a recorded maximum current gain of 134 at room temperature with a specific on‐resistance of 3.2 mΩ cm2 and a blocking voltage VCEO of 950 V. The SSR‐BJT kept a current gain of 60 °C at 250 °C with a specific on‐resistance of 8 mΩ cm2. These results have demonstrated the outstanding current‐gain capability of the SSR‐BJTs compared to conventional BJTs. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Hypersensitive response and acyl-homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae.

SummaryFire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.

Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Creation and genetic restoration of Erwinia amylovora strains with low levan synthesis

Erwinia amylovora synthesizes the enzyme levansucrase, which was found unprocessed after secretion. Its synthesis depends on several factors including rlsA, located in the hrp region and rlsB, located upstream of the levansucrase gene and a region downstream of lsc. A novel protein of 63 amino acids, RlsC, was identified as another regulator. Levan production of several natural levan-deficient strains and of an rlsC-mutant was strongly increased by expression of the cloned rlsC gene, but also of rlsA and rlsB. In addition, mutants in rlsA and rlsB were restored by overexpression of either one of the three Rls-proteins. A levan-deficient mutant downstream of lsc was only restored for levan synthesis by RlsA. In PCR assays with primers from an internal region of rlsA, a signal was obtained for Erwinia pyrifoliae and Erwinia strains from Japan, which do not produce levan, but not with rlsB or rlsC primers. Since only levansucrase expression but not the activity of the lsc-promoter was enhanced by RlsC and by RlsB, we assume interference of these factors on a post-transcriptional level.

Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv. oryzae.

We have cloned a hrp gene cluster from Xanthomonas oryzae pv. oryzae. Bacteria with mutations in the hrp region have reduced growth in rice leaves and lose the ability to elicit a hypersensitive response (HR) on the appropriate resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp portion of nucleotide sequence from the presumed left end and extending through the hrpB operon was determined. The region was most similar to hrp genes from Xanthomonas campestris pv. vesicatoria and Ralstonia solanacearum. Two new hrp-associated loci, named hpa1 and hpa2, were located beyond the hrpA operon. The hpa1 gene encoded a 13-kDa glycine-rich protein with a composition similar to those of harpins and PopA. The product of hpa2 was similar to lysozyme-like proteins. Perfect PIP boxes were present in the hrpB and hpa1 operons, while a variant PIP box was located upstream of hpa2. A strain with a deletion encompassing hpa1 and hpa2 had reduced pathogenicity and elicited a weak HR on nonhost and resistant host plants. Experiments using single mutations in hpa1 and hpa2 indicated that the loss of hpa1 was the principal cause of the reduced pathogenicity of the deletion strain. A 1,519-bp insertion element was located immediately downstream of hpa2. Hybridization with hpa2 indicated that the gene was present in all of the strains of Xanthomonas examined. Hybridization experiments with hpa1 and IS1114 indicated that these sequences were detectable in all strains of X. oryzae pv. oryzae and some other Xanthomonas species.

Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora

Natural Erwinia amylovora can display a reduced level of levan synthesis. A gene (rlsA) responsible for the suppression of low levansucrase synthesis by E. amylovora was cloned by complementation of natural levan-deficient strains lacking levansucrase expression. The rlsA region was sequenced, and an open reading frame of 266 amino acids was found. Sequence analysis revealed that RlsA resembles a transcriptional activator and may be a member of the LysR family. Overexpression of rlsA from a high copy number plasmid led to a two-fold increase of levan production, but had no influence on amylovoran synthesis. A mutant created by site-directed mutagenesis of wild type strain Eal/79 had the same phenotype as natural levan-deficient strains. The rlsA gene of natural levan deficient strains (with the exception of strain PD494) could be amplified by PCR, yielding the same DNA fragments as wild type strains. The gene was absent in a strain with a large deletion affecting the hrp region of E. amylovora . A match of the nucleotide sequence of rlsA with a sequence database revealed its identity with an incomplete open reading frame located downstream of two recently characterised dsp genes.

Isolation of hrp Cluster from Xanthomonas campestris pv. citri and Its Application for RFLP Analyses of Xanthomonads.

Two cosmid clones (pXCF13-38 and pXCF13-44) were isolated from a genomic library of Xanthomonas campestris pv. citri strain L-9 using pVir2, a cosmid clone of the hrp cluster of Ralstonia solanacearum, as the probe. 25kb region consisting on these clones was identified as the hrp region from a test of pathogenicity on citrus and of the hypersensitive response on tobacco after marker exchange of Tn3-Spice insertions. As pXCF13-38 covered almost the entire hrp cluster, this clone was used as the probe for RFLP analyses of xanthomonads. A dendrogram was drawn based on analyses of RFLP patterns and was distinguishable from reported dendrograms based on biochemical characters and on RFLP analyses using randomly chosen probes. From these results, the usefulness of RFLP analysis of hrp clusters for identification of various xanthmonads was shown and independent evolution of hrp cluster in xanthomonads was suggested.

DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way.

In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30 degrees C, and the highest expression was observed in M9 galactose minimal medium at 25 degrees C. A dsp::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative a factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA. A HrpL-dependent promoter sequence (GGAACC-N15-CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A sigma70 promoter sequence (TTGCCC-N16-GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL- background. Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.

Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes.

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains. KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.

Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction ofthe hypersensitive reaction.

The hrp cluster of the pepper and tomato pathogen Xanthomonas campestris pv. vesicatoria is required for both pathogenicity on susceptible host plants and induction of the hypersensitive reaction on resistant plants. The hrpA locus is located at the left end of the 25-kb hrp region and encodes a single 64-kDa Hrp protein, HrpA1, which belongs to the PulD superfamily of proteins involved in type II and type III protein secretion. In this study, we developed a defined medium without any plant-derived molecules that induces expression of hrpA in vitro. The hrpA transcription start site was mapped in the coding region of the hrpB8 gene, which is the last gene of the hrpB operon. The inducible hrpA promoter shows no homology to known promoter elements or other hrp loci of X. campestris pv. vesicatoria. hrpA expression was shown to be independent of the hrp regulatory gene hrpX. The amino acid sequence of the HrpA1 protein is predicted to contain an N-terminal signal sequence and no further transmembrane domains and to be rich in beta-sheet stretches. Expression of HrpA1 in Escherichia coli cells causes induction of the psp operon like some of its counterparts, suggesting some commonality of function and that HrpA1 forms multimers. The protein product of hrpA1 was identified by using a specific polyclonal antibody. Cell fractionation studies demonstrated that the HrpA1 protein is localized in the outer membrane of X. campestris pv. vesicatoria. HrpA1 is the first component of the Hrp secretion system whose localization has been determined in the original organism.

Organization of thehrpGene Cluster and Nucleotide Sequence of thehrpLGene fromPseudomonas syringaepv.morsprunorum

Pseudomonas syringae pv. morsprunorum PM7 is pathogenic to cherry and induces the hypersensitive response (HR) in tobacco. Six of 1,300 Tn5 mutants from strain PM7 neither elicit the HR in tobacco nor produce necrotic lesions in cherry plantlets. Plasmid pPM419, isolated from a genomic DNA library of wild-type strain PM7, restored the ability to cause a HR in five of the mutants. Restriction enzyme analysis of pPM419 revealed a 37-kilobase (kb) insert of genomic DNA from P. s. morsprunorum PM7. Tnt-spice mutagenesis of pPM419 and a second plasmid clone, pPM41, followed by marker exchange into the genome of P. s. morsprunorum PM7, indicated a 22-kb DNA fragment from P. s. morsprunorum PM7 has genes for elicitation of necrotic lesions in cherry plantlets and HR in tobacco. Complementation studies revealed that the hrp region of P. s. morsprunorum PM7 is organized into eight putative transcriptional units. Units II, VI, and VII exhibit DNA homology with the hrpI, hrpH, and hrpZ genes, respectively, of P. s. syringae 61. The nucleotide sequence of hrpL, the first transcriptional unit in the hrp cluster of P. s. morsprunorum PM7, encodes a polypeptide of 185 amino acids that exhibits 92% identity and 96% similarity with HrpL of P. s. syringae 61. Two transcriptional start sites, P1 and P2, located 63 and 25 bp upstream of the hrpL start codon, were identified by primer extension analysis. The −12 and −24 regions of the putative P2 promoter resemble a 54 consensus sequence

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.